Fibronectin based scaffold proteins

ABSTRACT

Fibronectin type III ( 10 Fn3) binding domains having novel designs that are associated with reduced immunogenicity are provided. The application describes alternative  10 Fn3 binding domains in which certain immunogenic regions are not modified when producing a binder in order to maintain recognition as a self antigen by the host organism. The application also describes  10 Fn3 binding domains in which HLA anchor regions have been destroyed thereby reducing the immunogenic contribution of the adjoining region. Also provided are  10 Fn3 domains having novel combinations of modified regions that can bind to a desired target with high affinity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/764,215, filed Jul. 29, 2015, which is a 35 U.S.C. 371 national stagefiling of International Application No. PCT/US2014/013784, filed Jan.30, 2014, which claims the benefit of U.S. Provisional Application No.61/759,639, filed Feb. 1, 2013.

SEQUENCE LISTING

The instant application contains a Sequence Listing, submitted herewith,which is hereby incorporated by reference in its entirety.

INTRODUCTION

Fibronectin is a large protein which plays essential roles in theformation of extracellular matrix and cell-cell interactions; itconsists of many repeats of three types (types I, II, and III) of smalldomains. Fibronectin type III domains (Fn3) are a large subfamily,members of which are frequently found as portions of cell adhesionmolecules, cell surface hormone and cytokine receptors, chaperones, andcarbohydrate-binding domains. For reviews see Bork & Doolittle, ProcNatl Acad Sci USA 89(19):8990-4 (1992); Bork et al., J Mol Biol.242(4):309-20 (1994); Campbell & Spitzfaden, Structure 2(5):333-7(1994); Harpez & Chothia, J Mol Biol. 238(4):528-39 (1994)).

Fibronectin based scaffolds are a family of proteins having animmunoglobulin like fold. These proteins, which generally make use of ascaffold derived from a fibronectin type III (Fn3) or Fn3-like domain,function in a manner characteristic of natural or engineered antibodies(that is, polyclonal, monoclonal, or single-chain antibodies) and, inaddition, possess structural advantages. Specifically, the structures ofthese antibody mimics have frequently been optimized for optimalfolding, stability, and solubility, even under conditions that normallylead to the loss of structure and function in antibodies. An example offibronectin-based scaffold proteins are Adnectins™ (Adnexus, a whollyowned subsidiary of Bristol-Myers Squibb). It has been shown that theCDR-like loop regions of the fibronectin based scaffolds can be modifiedto evolve a protein capable of binding to any compound of interest. Forexample, U.S. Pat. No. 7,115,396 describes Fn3 domain proteins whereinalterations to the BC, DE, and FG loops result in high affinity TNFαbinders. U.S. Pat. No. 7,858,739 describes Fn3 domain proteins whereinalterations to the BC, DE, and FG loops result in high affinity VEGFR2binders.

Protein pharmaceuticals are often associated with some degree ofimmunogenicity in a patient. These immunogenicity issues may result inreduction in efficacy of the protein therapeutic as well as potentiallyharmful immune responses in a patient. Accordingly, it would beadvantageous to obtain improved fibronectin domain scaffold proteinsthat are associated with reduced immunogenicity and that can be used forboth therapeutic and diagnostic purposes.

SUMMARY

One aspect of the application provides for fibronectin based scaffoldpolypeptides comprising novel combinations of modified loops andscaffold regions, e.g., the β-strands that are associated with improvedtarget binding. Another aspect of the application provides for novelfibronectin based scaffold polypeptides that are associated with reducedimmunogenicity.

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises (i) a modification in the amino acid sequence of at least onenorth pole loop selected from the BC, DE and FG loops relative to thecorresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or6), and (ii) a modification in the amino acid sequence of at least onesouth pole loop selected from the AB, CD and EF loops relative to thecorresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or6), wherein the at least one modified north pole loop and the at leastone modified south pole loop contribute to binding the same target. Insome embodiments, at least one of the north pole loops or at least oneof the south pole loops of the polypeptide has the amino acid sequenceof the corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ IDNO: 1 or 6).

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises (i) a modification in the amino acid sequence of at least oneof loops AB, BC, CD, DE, EF, or FG relative to the corresponding loop ofthe wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6), and (ii) amodification in the amino acid sequence of at least one β-strandrelative to the corresponding β-strand of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6), wherein the at least one modified loop andthe at least one modified β-strand contribute to binding the sametarget. In some embodiments, the polypeptides may comprise modifiedamino acid sequences in at least one β-strand and at least two loops. Insome embodiments, at least one modified loop of the polypeptide is anorth pole loop selected from the BC, DE and FG loops and at least onemodified loop is a south pole loop selected from the AB, CD and EF loopsand both loops contribute to binding to the target. In some embodiments,at least one loop is not modified, i.e., at least one loop has the aminoacid sequence of the corresponding loop of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6).

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises modifications in the amino acid sequences of the CD and FGloops relative to the sequences of the corresponding loops of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6), and wherein the CD andFG loops contribute to binding to the same target. In some embodiments,at least 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids of the CD loopare modified relative to the sequence of the CD loop of the wild-typehuman ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, one or moreamino acid residues of the CD loop corresponding to amino acid residues46 or 47 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6) are thesame as the wild-type amino acids at those positions. In someembodiments, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the aminoacids of the FG loop are modified relative to the sequence of the FGloop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In someembodiments, one or more amino acid residues of the FG loopcorresponding to amino acid residues 75 or 87 of the wild-type human¹⁰Fn3 domain (SEQ ID NO: 1 or 6) are the same as the wild-type aminoacids at those positions. In some embodiments, the amino acid sequenceof the CD loop, the FG loop, or both are extended in length or reducedin length relative to the amino acid sequence of the corresponding loopof the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). Combinationsthereof are also contemplated. For example, the amino acid sequence ofat least one of the CD and FG loops may be extended in length relativeto the corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ IDNO: 1 or 6) and the amino acid sequence of at least one of the CD and FGloops may be reduced in length relative to the corresponding loop of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments,polypeptides comprising ¹⁰Fn3 domains having modified CD and FG loops,may further comprise amino acid sequence modifications in one or more ofβ-strand C, and β-strand D, β-strand F and/or β-strand G relative to thesequences of the corresponding β-strands of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6). In some embodiments, the polypeptides havingmodified CD and FG loops may further comprise an amino acid sequencemodification in at least a portion of the BC loop, such as, for example,modifications in one or more amino acid residues of the BC loopcorresponding to amino acid residues 30 and 31 of the wild-type human¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, one or more ofthe modified β-strands, together with the modified loops, contribute tobinding to the same target. In some embodiments, one or more of the AB,DE and EF loops are not modified, i.e., the loops have the amino acidsequence of the corresponding loop of the wild-type human ¹⁰Fn3 domain(SEQ ID NO: 1 or 6).

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises modifications in the amino acid sequences of the CD and DEloops relative to the sequences of the corresponding loops of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6), and wherein the CD andDE loops contribute to binding to the same target. The polypeptide mayfurther comprise modifications in the amino acid sequences of one ormore of the EF loop, β-strand C, β-strand D, and/or β-strand F, and suchadditional modification may contribute to binding to the same targettogether with the CD and DE loops. In some embodiments, at least 10, 15,16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or all 31 of theresidues between the amino acids corresponding to residues 36 through 66of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6) have beenmodified relative to the corresponding residues in the wild-typesequence. In some embodiments, the CD loop is extended in length orreduced in length relative to the CD loop of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6).

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises modifications in the amino acid sequences of the EF and FGloops relative to the sequences of the corresponding loops of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6), and wherein the EF andFG loops contribute to binding to the same target. In some embodiments,the polypeptides may further comprise amino acid sequence modificationsin one or more of the AB loop, β-strand A and/or β-strand G, and suchadditional modifications may contribute to binding to the targettogether with the EF and FG loops. In some embodiments, the polypeptidesmay further comprise sequence modifications in the N-terminus and/orC-terminus. In particular, the amino acid sequence of the first 7 aminoacids or the amino acid sequence of the amino acids corresponding toresidues 93 through 97 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1or 6) may be modified relative to the corresponding residues in thewild-type sequence. These additional modifications at the termini mayalso contribute to binding to the target along with the other sequencemodifications. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or all 15 of the first 15 amino acid residues of the¹⁰Fn3 domain may be modified relative to the corresponding residues inthe wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In someembodiments, at least 3, 4 or 5 of the amino acid residues of the EFloop may be modified relative to the corresponding residues in thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments,at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or all18 of the residues between the amino acids corresponding to residues 80through 97 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6) maybe modified relative to the corresponding residues in the wild-typesequence. In some embodiments, the amino acid sequence of the FG loop isextended in length or reduced in length relative to the FG loop of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6).

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises modifications in the amino acid sequences of β-strand A, loopAB, β-strand B, loop CD, β-strand E, loop EF, and β-strand F relative tothe sequences of the corresponding 3-strands and loops of the wild-typehuman ¹⁰Fn3 domain (SEQ ID NO: 1 or 6), and wherein the modified loopsand strands contribute to binding to the same target. In someembodiments, the amino acid sequence of the CD loop is extended inlength or reduced in length relative to the CD loop of the wild-typehuman ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, thepolypeptides may further comprise a modification in the amino acidsequence of β-strand G and/or the C terminal tail.

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises a sequence modification in the FG loop relative to thesequence of amino acid residues 77-83 of loop FG of the wild-type human¹Fn3 domain (SEQ ID NO: 1 or 6), and wherein the ¹Fn3 binds to a targetwith a K_(d) of less than 500 nM. In some embodiments, the portion ofthe FG loop corresponding to amino acid residues 77-83 of the wild-typehuman ¹Fn3 domain (SEQ ID NO: 1 or 6) is extended in length or reducedin length relative to the sequence of amino acid residues 77-83 of thewild-type FG loop. In some embodiments, the FG loop alone mediatesbinding to the target. In some embodiments, one of more of the AB, BC,CD, DE or EF loops has the sequence of the corresponding loop of thewild-type human ¹Fn3 domain (SEQ ID NO: 1 or 6).

In some embodiments, the polypeptides provided herein comprise a humanfibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises a sequence modification in the BC loop and at least one ofβ-strand B or β-strand C relative to the sequences of the correspondingloop and β-strands of the wild-type human ¹Fn3 domain (SEQ ID NO: 1 or6), and wherein the ¹⁰Fn3 domain has reduced immunogenicity relative toan equivalent ¹⁰Fn3 domain that does not have a sequence modification inat least one of β-strand B or β-strand C relative to wild-type. In someembodiments, the amino acid sequence of the BC loop is extended inlength or reduced in length relative to the amino acid sequence of theBC loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In someembodiments, the ¹⁰Fn3 domain further comprises a modification in theamino acid sequence of the first 7 amino acid residues relative to theamino acid sequence of the first 7 amino acid residues of the wild-typehuman ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, the ¹⁰Fn3domain further comprises a modification in the DE loop, the FG loop, orboth, relative to the sequences of the corresponding loops of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments,the amino acid sequence of the DE loop is extended in length or reducedin length relative to the amino acid sequence of the DE loop of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments,the amino acid sequence of the FG loop is extended in length or reducedin length relative to the amino acid sequence of the FG loop of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments,the ¹⁰Fn3 domain comprises a sequence modification in the DE loop, andfurther comprises a sequence modification in β-strand D, β-strand E, orboth, relative to the sequences of the corresponding β-strands of thewild-type human ¹Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments,the ¹Fn3 domain comprises a sequence modification in the FG loop, andfurther comprises a sequence modification in β-strand F, β-strand G, orboth, relative to the sequences of the corresponding β-strands of thewild-type human ¹Fn3 domain (SEQ ID NO: 1 or 6).

In some embodiments, at least a portion of the BC loop of thepolypeptides provided herein have the amino acid sequence of thecorresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or6). For example, the first 1, 2, 3, 4, 5, 6, 7 or 8 residues of the BCloop may be the same as the corresponding residues in the BC loop of thewild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In still otherembodiments, the entire BC loop has the amino acid sequence of thecorresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or6). In some embodiments, polypeptides having at least a portion of theBC loop with the wild-type sequence have reduced immunogenicity relativeto an equivalent polypeptide having additional modifications in the BCloop.

In particular embodiments, the polypeptides provided herein comprise ahuman fibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises a sequence modification in a portion of the BC loop and aportion of the FG loop relative to the sequence of the correspondingloops of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6), andwherein the ¹⁰Fn3 domain has reduced immunogenicity relative to anequivalent ¹⁰Fn3 domain having a greater portion of the BC loop modifiedrelative to the wild-type BC loop. In some embodiments, the portion ofthe BC loop that is modified may correspond to residues 28-29, 27-29,26-29, 25-29, or 24-29 of the BC loop of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6). In some embodiments, the portion of the FGloop that is modified may corresponds to residues 77-79, 77-80, 77-81,77-82, 77-83, 77-84, 77-85, or 77-86 of the FG loop of the wild-typehuman ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, the portionof the FG loop that is modified has an insertion or deletion relative tothe corresponding portion of the FG loop of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6). In some embodiments, the BC and FG loopcontribute to binding to the target. In some embodiments, the ¹⁰Fn3domain further comprises a sequence modification in a portion of the DEloop relative to the sequence of the DE loop of the wild-type human¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, the portion ofthe DE loop that is modified corresponds to residues 52 and 53 of the BCloop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1). In someembodiments, the ¹⁰Fn3 domain has reduced immunogenicity relative to anequivalent ¹⁰Fn3 domain further comprising modifications in one or moreof amino acid residues 23-27, relative to the corresponding positions inthe wild-type BC loop. In some embodiments, the ¹⁰Fn3 domain binds to atarget with a K_(d) of less than 500 nM.

In some embodiments, the FG loop of the polypeptides provided hereindoes not contain an RGD integrin binding site.

In some embodiments, the hydrophobic core residues of the polypeptidesprovided herein have not been modified relative to the wild-type human¹⁰Fn3 domain (SEQ ID NO: 1 or 6).

In some embodiments, the amino acid sequence of at least one of themodified loops of the polypeptides provided herein has been extended inlength relative to the amino acid sequence of the corresponding loop ofthe wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). In otherembodiments, the amino acid sequence of at least one of the modifiedloops has been reduced in length relative to the amino acid sequence ofthe corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1or 6).

In some embodiments, the amino acid sequence of the C terminal tail ofthe polypeptides provided herein is modified relative to the amino acidsequence of the C-terminal tail of the wild-type human ¹⁰Fn3 domain (SEQID NO: 1 or 6). In some embodiments, the amino acid sequence of thefirst 7 amino acid residues is modified relative to the amino acidsequence of the first 7 amino acid residues of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6). In other embodiments, the polypeptide hasfrom 1-7 amino acids truncated from the N-terminus relative to thewild-type human ° Fn3 domain (SEQ ID NO: 1 or 6), from 1-9 amino acidstruncated from the C-terminus relative to the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1), or both.

In some embodiments, the polypeptides provided herein have at least 50%identity to the amino acid sequence of the wild-type human ¹⁰Fn3 domain(SEQ ID NO: 1, 2, 60 or 6). In other embodiments, the polypeptide has atleast 65% identity to the amino acid sequence of the wild-type human¹⁰Fn3 domain (SEQ ID NO: 1, 2, 60 or 6). In certain embodiments, the¹⁰Fn3 domains comprise an amino acid sequence that is at least 60, 70,80 or 90% identical to the naturally occurring human ¹⁰Fn3 domainrepresented by SEQ ID NO: 1, 2, 60 or 6.

In some embodiments, the polypeptides provided herein comprise afibronectin type III tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence having at least 60% identity to SEQ IDNO: 2 or 60 and binds to a target molecule with a K_(d) of less than 100nM, and wherein the ¹⁰Fn3 domain further comprises a C-terminal tailthat does not contain a DK sequence. In some embodiments, the C-terminaltail comprises the amino acid sequence of SEQ ID NO: 7. In someembodiments, the C-terminal tail further comprises a cysteine residue.In other embodiments, the C-terminal tail comprises the sequence of SEQID NO:8. In other embodiments, the C-terminal tail may comprise thesequence of any one of SEQ ID NOs: 23-31.

In certain embodiments, the fibronectin based scaffold proteins bind toa target that is not bound by a wild-type ¹⁰Fn3 domain.

In some embodiments, the ¹⁰Fn3 domains of the fibronectin based scaffoldprotein further comprises an N-terminal extension comprising from 1-10amino acids. In certain embodiments, the ¹⁰Fn3 domain comprises an M, MGor G N-terminal to first amino acid of SEQ ID NO: 1 or 6. In otherembodiments, the amino acid residues corresponding to amino acids 1-8 ofSEQ ID NO: 1 or 6 are replaced with any one of SEQ ID NOs: 9-11 or16-21.

In some embodiments, the fibronectin based scaffold proteins furthercomprise one or more pharmacokinetic (PK) moieties selected from: apolyoxyalkylene moiety, a human serum albumin binding protein, sialicacid, human serum albumin, transferrin, IgG, an IgG binding protein, andan Fc fragment. In some embodiments, the PK moiety is thepolyoxyalkylene moiety and said polyoxyalkylene moiety is polyethyleneglycol (PEG). In some embodiments, the PEG moiety is covalently linkedto the fibronectin based scaffold protein via a Cys or Lys amino acid.In some embodiments, the PEG is between about 0.5 kDa and about 100 kDa.

In certain embodiments, the application provides pharmaceuticallyacceptable compositions comprising the novel ¹⁰Fn3 domains describedherein. In some embodiments, the composition is essentially pyrogenfree. In some embodiments, the composition is substantially free ofmicrobial contamination making it suitable for in vivo administration.The composition may be formulated, for example, for intravenous (IV),intraperiotoneal (IP) or subcutaneous (SubQ) administration. In someembodiments, the composition comprises a physiologically acceptablecarrier. In some embodiments, the pH of the composition is between4.0-6.5, between 4.0-5.5, or is equal to 4.0, 4.5, 5.0 or 5.5. In someembodiments, the concentration of the fibronectin based scaffold proteinis 5 mg/ml in the composition.

In certain embodiments, the application provides a nucleic acid encodingthe novel ¹⁰Fn3 domains as described herein. Vectors containingpolynucleotides for such proteins are included as well. Suitable vectorsinclude, for example, expression vectors. A further aspect of theapplication provides for a cell, comprising a polynucleotide, vector, orexpression vector, encoding a ¹⁰Fn3 domain. Sequences are preferablyoptimized to maximize expression in the cell type used. In someembodiments, expression is in a bacterial cell, such as E. coli. Inother embodiments, expression is in a mammalian cell. In one embodiment,the cell expresses a protein comprising a ¹⁰Fn3 domain as describedherein. In certain embodiments, the polynucleotides encoding a ¹⁰Fn3domain are codon optimized for expression in the selected cell type.Also provided are methods for producing a ¹⁰Fn3 domain as describedherein, comprising culturing a host cell comprising a nucleic acid,vector, or expression vector encoding a ¹⁰Fn3 domain and recovering theexpressed protein from the culture.

In certain embodiments, the application provides libraries of thefibronectin based scaffold proteins described herein. The librariesprovided herein may comprise, for example, at least 10⁵, 10⁶, 10⁷, 108,10⁹, 10¹⁰, 10¹², 10¹³, or 10¹⁴, or more members. Also provided aremethods for isolating a fibronectin based scaffold protein thatspecifically binds to a target of interest from one of the librariesdescribed herein. For example, a library isolation method may comprise,for example, contacting a library of fibronectin based scaffold proteinswith a target of interest, and isolating members of the library thatbind to the target (e.g., with a particular affinity or under suitablewash conditions). The isolation step may be carried out using anysuitable method, such as phage display or mRNA display. Similarly,target binding may be conducted using any suitable method such asimmobilizing the target on a solid support (e.g., a column, chip, bead,etc.) and mixing the immobilized target with the library underconditions suitable to allow protein binding. The bound library membersmay then be separated from unbound library members to yield an isolatedfibronectin based scaffold protein that binds to the target. In certainembodiments, the isolation method may involve repeated rounds of targetbinding and isolation steps.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The wildtype ¹⁰Fn3 amino acid sequence (SEQ ID NO: 1) in whichthe hydrophobic core amino acid residues are indicated. The beta-strandsare in bold, loop regions are designated by letter pairs, and thehydrophobic core residues are underlined in bold. Amino acids 95-101correspond to a tail, which when absent from a ¹⁰Fn3 molecule having SEQID NO: 1 forms a 10Fn3 molecule having SEQ ID NO: 6.

FIGS. 2A-2F: Wildtype 10Fn3 amino acid sequences (SEQ ID NO: 1 or 6) inwhich amino acid positions that may be mutated to provide arepresentative modified 10Fn3 polypeptide patch library are indicated inthe full-length sequence (SEQ ID NO: 1). Potential positions that may bemodified to generate each of the respective classes of patch library10Fn3 polypeptides are in bold and underlined. Any one of, orcombination of, the positions indicated may be mutated to generateeither Northwest Binders (FIG. 2A), Northeast Binders (FIG. 2B), WestSide Binders (FIG. 2C), South-Front Binders (FIG. 2D), AG Strand Binders(FIG. 2E) and South West Binders (FIG. 2F).

FIGS. 3A-3F: Crystal structures of the wildtype 10Fn3 domain, in whichviews of different possible binding interfaces are shown. Residues thatmay be varied from wild-type are shown in black. Sticks are added tovaried residues that are not members of one of the six loops. Crystalstructure views of the Northwest binding interface (FIG. 3A), Northeastbinding interface (FIG. 3B), West Side binding interface (FIG. 3C),South-Front binding interface (FIG. 3D), AG Strand binding interface(FIG. 3E) and South West binding interface (FIG. 3F) of the wildtype10Fn3 domain are shown.

FIG. 4: HLA-binding data showing the IC₅₀ binding affinity (μM) of fivedifferent HLA allele proteins to five different peptide segments of the¹⁰Fn3 polypeptide. SEQ ID NOs: 58, 51 and 52 are loop region clusters ofthe BC, DE and FG loops, respectively, with loop region residuesunderlined. SEQ ID NOs: 53 and 54 are a wildtype and modified scaffoldregion segment of the ¹⁰Fn3 polypeptide, respectively. As indicated, theBC loop region cluster (SEQ ID NO: 58) and the two scaffold regionpeptide segments tested (SEQ ID NOs: 53 and 54) were strong binders (<25μM) of most of the HLA allele proteins tested. The predictedimmunodominant regions of the scaffold region peptide segments (SEQ IDNOs: 53 and 54) are underlined.

FIG. 5: Notch 1:murine DLL4 competition assay results showing that theWS-LI1 binders of SEQ ID NOs: 3 and 4 were capable of 100% inhibition,and that the WS-LI1 binder of SEQ ID NO: 5 was capable of 75%inhibition, of the interaction between Notch 1 and murine DLL4, asdetermined by Biacore analysis.

FIG. 6: Size Exclusion Chromatography results showing that the WS-LI1binder of SEQ ID NO: 3 was predominantly monomeric, while the WS-LI1binders of SEQ ID NOs: 4 and 5 contained a mixture of monomeric andaggregated proteins. Bold traces correspond to the WS-LI1 binder tested,and non-bold traces correspond to molecular weight markers, with theexpected elution of the WS-LI1 binder monomers to be eluted between the3^(rd) and 5^(th) marker peaks.

FIG. 7: Size Exclusion Chromatography results showing that the WS-LI1binders of SEQ ID NOs: 45-47 were predominantly monomeric. Bold tracescorrespond to the WS-LI1 binders tested, and non-bold traces correspondto molecular weight markers, with the expected elution of the WS-LI1binder monomers to be eluted between the 3^(rd) and 5^(th) marker peaks.

FIG. 8: Size Exclusion Chromatography results showing that the WS1binders of SEQ ID NOs: 48-49 were predominantly monomeric. Bold tracescorrespond to the WS1 binders tested, and non-bold traces correspond tomolecular weight markers, with the expected elution of the WS1 bindermonomers to be eluted between the 3^(rd) and 5^(th) marker peaks.

FIGS. 9A-9C: Library designs for non-traditional 10Fn3 binders. FIG. 9Ashows library designs in which the BC loop is substantially orcompletely unmodified (i.e., all or most of the BC loop is left as thewild-type sequence). All of the CD and FG loops of the librariesdepicted in FIG. 9A may be varied in size, in particular, loop CD ofSP1, WS2′, WS2′-CD, Front3 and CD1-loop may be varied in length. Loop FGof Back3 may also be varied in size, and the first three amino acids ofCD1-loop (i.e., VSD) may be deleted. FIG. 9B shows library designs inwhich varying portions of the N-terminus of the BC loop have been leftas wild-type. FIG. 9C shows library designs in which the BC loop and oneor both of the B-strand regions flanking the BC loop are modified.Library NP-6 may also be constructed by keeping the threonine atposition 71 constant and/or by keeping the length of amino acids 1-7constant. Library NP4-5 may also be constructed by keeping the threonineat position 71 constant, and/or by keeping the length of amino acids 1-7constants, and/or by keeping loop BC constant. In each of FIGS. 9A-9C,the full-length wild-type 10Fn3 domain (SEQ ID NO: 1) is shown at thetop with numbering from amino acid 1 to amino acid 101 and markingsindicating the loop and strand regions. Below the depiction of the 10Fn3wild-type domain is a depiction of a classic north pole library design(i.e., with the BC, DE and FG loops modified). The non-traditionallibrary designs are shown below the classic north pole library design.Positions that may be modified by substitution are indicated in bold andunderlined, regions that may be modified by substitution, insertionand/or deletion are in bold and boxed, and amino acid residues that arenon-wild-type are shaded. In FIG. 9B, all sequences are based on SEQ IDNO: 1. In FIG. 9C, the WT 10Fn3, Classic NP, NP1, NE1, NP4-5, NP6-1, andNW2 sequences are disclosed as SEQ ID NO: 1; and all remaining sequencesare disclosed as SEQ ID NO: 59. In FIG. 9A, the WT 10Fn3, Classic NP,WS1, WS2, WS3, LI-3(a), WS-LI1, WS2′ and WS2′-CD are based on SEQ ID NO:1; and all remaining sequences are based on SEQ ID NO: 59. All librariesmay also be based on SEQ ID NO: 6 or SEQ ID NO: 12, i.e., lacking aminoacids 95-101 of SEQ ID NOS 1 and 59, respectively.

FIG. 10: Sequence alignment of 8 ¹⁰Fn3 polypeptides (Adnectin-1 toAdnectin-8), which bind specifically to human PXR ligand binding domain,with the parent ¹⁰Fn3 domain (SEQ ID NO: 1). The location of theβ-strands is indicated by the arrows below the sequence alignment, withcorresponding amino acids indicated in bold. Adnectins-3 (SEQ ID NO: 62)and -4 (SEQ ID NO: 63) correspond to SEQ ID NOs: 48 and 49 with anadditional 6xHis tail (SEQ ID NO: 44). Adnectins-1, -2, -5, -6, -7 and-8 (SEQ ID NOs: 70-72 and 13-15, respectively) correspond to SEQ ID NOs:64-69, respectively, with an additional 6xHis tail (SEQ ID NO: 44).

FIG. 11: Histogram showing the degree of binding of (from left to right)vitronectin, fibronectin, a non-binding control adnectin (with RGDchanged to RGE), and three different ¹⁰Fn3 molecules binding to aspecific target and not comprising an RGD sequence (¹⁰Fn3 A, B and C,respectively) to immobilized integrin AlphaV-Beta3.

FIG. 12: Amino acid sequences of wild-type human ¹⁰Fn3 (SEQ ID NO: 6)(top line) and those of libraries WS4, WS5, WS6 and WS7. The underlinedbolded and boxed amino acids can be changed by substitution, deletionand addition. The underlined amino acids can be changed by substitution.

FIGS. 13A-13C: Amino acid sequence of 10Fn3 molecules (SEQ ID NOs:73-131), wherein x is any amino acid or x is any amino acid except theamino acid at the corresponding position in the wild-type 10Fn3 molecule(SEQ ID NO: 1 or 6). The subscript after “x” indicates the number ofamino acid residues. For example, “x3-5” refers to 3 to 5 amino acidresidues.

FIGS. 14A-14E: Amino acid sequences of exemplary 10FN3 molecules havingdesigns shown in FIG. 13, or differing therefrom in at most a few, e.g.,1, 2, 3, 4 or 5 amino acids.

DETAILED DESCRIPTION Definitions

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

A “region” of a ¹⁰Fn3 domain as used herein refers to either a loop (AB,BC, CD, DE, EF and FG), a β-strand (A, B, C, D, E, F and G), theN-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO: 1),or the C-terminus (corresponding to amino acid residues 93-101 of SEQ IDNO: 1) of the human ¹⁰Fn3 domain.

A “north pole loop” refers to any one of the BC, DE and FG loops of afibronectin human fibronectin type 3 tenth (¹⁰Fn3) domain.

A “south pole loop” refers to any one of the AB, CD and EF loops of afibronectin human fibronectin type 3 tenth (¹⁰Fn3) domain.

A “scaffold region” refers to any non-loop region of a human ¹⁰Fn3domain. The scaffold region includes the A, B, C, D, E, F and Gβ-strands as well as the N-terminal region (amino acids corresponding toresidues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acidscorresponding to residues 93-101 of SEQ ID NO: 1).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

As used herein, an amino acid residue in a polypeptide is considered to“contribute to binding” a target if (1) any of the non-hydrogen atoms ofthe residue's side chain or main chain is found to be within fiveangstroms of any atom of the binding target based on an experimentallydetermined three-dimensional structure of the complex, and/or (2)mutation of the residue to its equivalent in wild-type ¹⁰Fn3 (e.g., SEQID NO: 1 or 6), to alanine, or to a residue having a similarly sized orsmaller side chain than the residue in question, leads to a measuredincrease of the equilibrium dissociation constant to the target (e.g.,an increase in the k_(on)).

The “half-life” of a polypeptide can generally be defined as the timetaken for the serum concentration of the polypeptide to be reduced by50%, in vivo, for example due to degradation of the polypeptide and/orclearance or sequestration of the polypeptide by natural mechanisms. Thehalf-life can be determined in any manner known per se, such as bypharmacokinetic analysis. Suitable techniques will be clear to theperson skilled in the art, and may, for example, generally involve thesteps of administering a suitable dose of a polypeptide to a primate;collecting blood samples or other samples from said primate at regularintervals; determining the level or concentration of the polypeptide insaid blood sample; and calculating, from (a plot of) the data thusobtained, the time until the level or concentration of the polypeptidehas been reduced by 50% compared to the initial level upon dosing.Methods for determining half-life may be found, for example, in Kennethet al., Chemical Stability of Pharmaceuticals: A Handbook forPharmacists (1986); Peters et al, Pharmacokinete analysis: A PracticalApproach (1996); and “Pharmacokinetics”, M Gibaldi & D Perron, publishedby Marcel Dekker, 2nd Rev. edition (1982).

Half-life can be expressed using parameters such as the t1/2-alpha,t1/2-beta and the area under the curve (AUC). In the presentspecification, an “increase in half-life” refers to an increase in anyone of these parameters, any two of these parameters, or in all threethese parameters. In certain embodiments, an increase in half-liferefers to an increase in the t1/2-beta, either with or without anincrease in the t1/2-alpha and/or the AUC or both.

Overview

¹⁰Fn3 domains are structurally and functionally analogous to antibodies,specifically the variable region of an antibody. Historically, designsof ¹Fn3 binding domains having relied on the similarity of the ¹⁰Fn3domain structure to that of the VH domain of an antibody. In particular,¹⁰Fn3 binding domains traditionally have relied on modifications in theamino acid sequences of the CDR-like loops of the ¹⁰Fn3 domain. Each ofthe AB, BC, CD, DE, EF and FG loops of ¹⁰Fn3 domains is analogous to acomplementary determining region (CDR) from an immunoglobulin in thatthey are flexible and amenable to modifications in their amino acidsequences without altering the overall structure of the ¹⁰Fn3 domain.Furthermore, modifications of sets of the CDR-like loops along one faceof the ¹⁰Fn3 domain (i.e., the “north pole”) have been shown to permitdevelopment of ¹⁰Fn3 domains that binds to a desired target (see e.g.,PCT Publication Nos. WO 02/032925, WO 2008/097497, and No. WO2008/066752). In these traditional ¹⁰Fn3 scaffold designs, the proteinsequences between the loops, i.e. the β-strands, are typically notmodified or are only minimally modified because they play a role inmaintaining the overall structural conformation of the ¹⁰Fn3. We havenow surprisingly found that it is possible to modify the ¹⁰Fn3 domainsin a non-traditional manner to produce proteins that bind to a desiredtarget while maintaining suitable stability.

In particular, the present application provides fibronectin basedscaffold polypeptides comprising novel combinations of modified loopsand scaffold regions and that are associated with improved properties.The fibronectin based scaffold proteins described herein comprise one ormore human tenth fibronectin type III domains that have been modified soas to bind to one or more desired targets. The present applicationrelates, in part, to the surprising discovery that novel combinations offibronectin domain loop and/or scaffold region modifications areassociated with specific target binding. In particular, it has beendiscovered that novel scaffold region, e.g. non-loop, modifications infibronectin-based scaffold proteins may be combined with specific loopmodifications to obtain specific target binding. Such novel scaffolddesigns provide expanded potential for designing ¹⁰Fn3 based bindingproteins. For example, the non-traditional scaffold designs describedherein permit the creation of libraries with greater diversity byopening up new areas for sequence modification within the ¹⁰Fn3 domain.In addition, the non-traditional scaffold designs allow for alternativesurface interface geometries as compared to the interface geometriesprovided by the traditional CDR-like loop interface. The additionaldiversity and alternative surface geometries provided by thesenon-traditional binders may facilitate development of ¹⁰Fn3 bindingdomains with desirable properties, for example, by providing ¹⁰Fn3binding domains with higher affinity for a given target, or by providing¹⁰Fn3 binding domains that bind to different epitopes on a given target.

The application also describes novel fibronectin based scaffoldpolypeptides that are associated with reduced immunogenicity. Asdescribed in the examples herein, it has been discovered the β-strandB/BC loop/β-strand C region may be an immunogenic ‘hot spot’ based onstrong HLA binding activity. In particular, this region appears to serveas a strong anchor sequence for HLA binding. The examples also show thatthe wild-type sequence for the β-strand B/BC loop/β-strand C region isrecognized as a self-antigen by a primate host. Therefore, despitestrong HLA binding, no immune response is generated to the wild-typesequence. Accordingly, we have developed alternative ¹⁰Fn3 scaffolds inwhich the key areas within the β-strand B/BC loop/β-strand C region havebeen left as wild-type, while modifications in other regions of thesequence permit high affinity target binding. Such alternative binderswill have an increased chance of generating high affinity ¹⁰Fn3 bindingdomains that avoid undesirable immune responses in a host organismbecause the β-strand B/BC loop/β-strand C region immunogenic hot spot isunaltered and therefore should be recognized as a self-antigen by thehost organism. The application also provides alternative ¹⁰Fn3 bindingdomains in which the HLA anchor sequence in the β-strand B/BCloop/β-strand C region has been destroyed, which should reduce theimmunogenic potential of this region. Such ¹⁰Fn3 binding domains withthe anchor sequence removed should allow diversification of all or aportion of the BC loop, while still avoiding undesirable immuneresponses associated with this region. The HLA anchor sequence can beremoved or destroyed by modifying key residues in the β-strand B and/orβ-strand C regions, in conjunction with modifications to the BC loopregion. Exemplary non-traditional ¹⁰Fn3 binding domains having reducedimmunogenic potential are described further below.

The novel fibronectin based scaffold polypeptides described herein maybe designed to bind to any target of interest. In exemplary embodiments,the target is an antigen, a polypeptide or a therapeutic protein targetof interest. Exemplary therapeutically desirable targets, include, forexample, tumor necrosis factor alpha (TNF-alpha), delta-like protein 4(DLL4), interleukin 17 (IL-17), and pregnane X receptor (PXR).

Fibronectin Based Scaffolds

A. General Structure

Fn3 refers to a type III domain from fibronectin. An Fn3 domain issmall, monomeric, soluble, and stable. It lacks disulfide bonds and,therefore, is stable under reducing conditions. The overall structure ofFn3 resembles the immunoglobulin fold. Fn3 domains comprise, in orderfrom N-terminus to C-terminus, a beta or beta-like strand, A; a loop,AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-likestrand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a betaor beta-like strand, E; a loop, EF; a beta or beta-like strand, F; aloop, FG; and a beta or beta-like strand, G. The seven antiparallel3-strands are arranged as two beta sheets that form a stable core, whilecreating two “faces” composed of the loops that connect the beta orbeta-like strands. Loops AB, CD, and EF are located at one face (“thesouth pole”) and loops BC, DE, and FG are located on the opposing face(“the north pole”). Any or all of loops AB, BC, CD, DE, EF and FG mayparticipate in ligand binding. There are at least 15 different Fn3modules in human Fibronectin, and while the sequence homology betweenthe modules is low, they all share a high similarity in tertiarystructure.

In exemplary embodiments, the ligand binding scaffold proteins describedherein are based on the tenth fibronectin type III domain, i.e., thetenth module of Fn3, (¹⁰Fn3). The amino acid sequence of a naturallyoccurring human ¹⁰Fn3 is set forth in SEQ ID NO: 1:

(SEQ ID NO: 1) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSQ(the AB, CD and EF loops are underlined; the BC, FG, and DE loops areemphasized in bold; the β-strands are located between each of the loopregions; and the N-terminal and C-terminal regions are shown initalics). SEQ ID NO: 1 is the sequence of a ¹⁰Fn3 molecule thatcomprises a tail, i.e., amino acids 95-101. SEQ ID NO: 6 is the aminoacid sequence of a wild-type human ¹⁰Fn3 molecule that does not comprisea tail and consists of amino acids 1-94 of SEQ ID NO: 1.

Residues involved in forming the hydrophobic core (the “core amino acidresidues”) in SEQ ID NO: 1 include the amino acids corresponding to thefollowing amino acids of SEQ ID NO: 1 or 6: L8, V10, A13, L18, I20, W22,Y32, I34, Y36, F48, V50, A57, I59, L62, Y68, I70, V72, A74, I88, I90 andY92, wherein the core amino acid residues are represented by the singleletter amino acid code followed by the position at which they arelocated within SEQ ID NO: 1. See e.g., Dickinson et al., J. Mol. Biol.236: 1079-1092 (1994). In some embodiments, the residues involved informing the hydrophobic core are used to determine the boundaries of theloop regions of the polypeptide. For example, the AB loop may be definedas being the stretch of amino acids between the hydrophobic core residueA13 of β-strand A and the hydrophobic core residue L18 of the β-strandB. See FIG. 1. In some embodiments, the hydrophobic core amino acids arenot modified relative to the wild-type sequence. In other embodiments,the following hydrophobic amino acids may be mutated: A13, which is partof a beta bulge and can convert to a surface residue; Y32 and A74,either or both of which can vary to interact differently with the nearbyloops; 188, which is partially solvent-exposed and the correspondingposition is not always a hydrophobic residue in natural fibronectin typeIII domains; and Y92, which connects to the C-terminal tail and could bediversified if the C-terminal region is diversified.

In some embodiments, the AB loop corresponds to residues 14-17, the BCloop corresponds to residues 23-31, the CD loop corresponds to residues37-47, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 63-67, and the FG loop corresponds to residues75-87 of SEQ ID NO: 1. The BC, DE and FG loops align along one face ofthe molecule, i.e. the “north pole”, and the AB, CD and EF loops alignalong the opposite face of the molecule, i.e. the “south pole”. In SEQID NO: 1, β-strand A corresponds to residues 8-13, β-strand Bcorresponds to residues 18-22, β-strand C corresponds to residues 32-36,beta strand D corresponds to residues 48-50, β-strand E corresponds toresidues 57-62, β-strand F corresponds to residues 68-74, and β-strand Gcorresponds to residues 88-92. The β-strands are connected to each otherthrough the corresponding loop, e.g., strands A and B are connected vialoop AB in the formation β-strand A, loop AB, β-strand B, etc. TheN-terminal and/or C-terminal regions (italicized above), may be removedor altered to generate a molecule retaining biological activity andcomprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. Incertain embodiments, the first 8 amino acid residues of SEQ ID NO: 1and/or the last 7 amino acid residues of SEQ ID NO: 1 (i.e., amino acidresidues 95-101 of SEQ ID NO: 1) may be removed or altered to generate apolypeptide comprising the amino acid sequence of SEQ ID NO: 60 or SEQID NO: 6 (corresponding to SEQ ID NO: 1 without the 7 N-terminal aminoacids, and consisting of amino acids 1-94 of SEQ ID NO: 1). Thelibraries described herein may comprise the N- or C-terminal region setforth in SEQ ID NO: 1. In certain embodiments, the libraries comprisethe N-terminal region, but do not comprise the C-terminal region (i.e.,they are based on SEQ ID NO: 6).

As described above, amino acid residues corresponding to residues 14-17,23-31, 37-47, 51-56, 63-67 and 75-87 of SEQ ID NO: 1 define the AB, BC,CD, DE, EF and FG loops, respectively. However, it should be understoodthat not every residue within a loop region needs to be modified inorder to achieve a ¹⁰Fn3 binding domain having strong affinity for adesired target. For example, in some embodiments, only residuescorresponding to amino acids 39-45 of the CD loop and 77-87 of the FGloop were modified to produce high affinity ¹⁰Fn3 binders (see e.g., themurine DLL4 binding cores having an amino acid sequence of either SEQ IDNO: 3, 4 or 5 and the murine IL-17 binding cores having an amino acidsequence of either SEQ ID NO: 45, 46 or 47).

Additionally, insertions and deletions in the loop regions may also bemade while still producing high affinity ¹⁰Fn3 binding domains. Forexample, the CD loop of the murine DLL4 binder having SEQ ID NO: 3 hasthe same length CD loop as the wild-type ¹⁰Fn3 domain, i.e., the sevenresidues 39-45 of SEQ ID NO: 1 were replaced with the seven residues41-47 of SEQ ID NO: 3. In contrast, the FG loop of the murine DLL4binder having SEQ ID NO: 3 is longer in length than the corresponding FGloop of the wild-type ¹⁰Fn3 domain, i.e., the nine residues 77-85 of SEQID NO: 1 were replaced with the nineteen residues 79-98 of SEQ ID NO: 3.

Accordingly, in some embodiments, one or more loops selected from AB,BC, CD, DE, EF and FG may be extended or shortened in length relative tothe corresponding loop in wild-type human ¹⁰Fn3. In any givenpolypeptide, one or more loops may be extended in length, one or moreloops may be reduced in length, or combinations thereof. In someembodiments, the length of a given loop may be extended by 2-25, 2-20,2-15, 2-10, 2-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, or 10-15 aminoacids. In some embodiments, the length of a given loop may be reduced by1-15, 1-11, 1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids. Inparticular, the FG loop of ¹⁰Fn3 is 13 residues long, whereas thecorresponding loop in antibody heavy chains ranges from 4-28 residues.To optimize antigen binding in polypeptides relying on the FG for targetbinding, therefore, the length of the FG loop of ¹⁰Fn3 may be altered inlength as well as in sequence to obtain the greatest possibleflexibility and affinity in target binding.

In some embodiments, one or more residues of the integrin-binding motif“arginine-glycine-aspartic acid” (RGD) (amino acids 78-80 of SEQ ID NO:1 or 6) may be substituted so as to disrupt integrin binding. In someembodiments, the FG loop of the polypeptides provided herein does notcontain an RGD integrin binding site. In one embodiment, the RGDsequence is replaced by a polar amino acid-neutral amino acid-acidicamino acid sequence (in the N-terminal to C-terminal direction). Inanother embodiment, the RGD sequence is replaced with SGE. In yetanother embodiment, the RGD sequence is replaced with RGE.

In some embodiments, a fibronectin based scaffold protein comprises a¹⁰Fn3 domain having at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or 90% identity to the human ¹Fn3 domain having the amino acidsequence of SEQ ID NO: 1, 2, 60 or 6. In certain embodiments, thepolypeptides provided herein have at least 50% identity to the aminoacid sequence of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1, 2, 60or 6). In other embodiments, the polypeptide has at least 65% identityto the amino acid sequence of the wild-type human ¹⁰Fn3 domain (SEQ IDNO: 1, 2, 60 or 6). In certain embodiments, one or more of the loopswill not be modified relative to the sequence of the corresponding loopof the wild-type sequence and/or one or more of the β-strands will notbe modified relative to the sequence of the corresponding β-strand ofthe wild-type sequence. In certain embodiments, each of the beta orbeta-like strands of a ¹Fn3 domain in a fibronectin based scaffoldprotein may comprise, consist essentially of, or consist of an aminoacid sequence that is at least 80%, 85%, 90%, 95% or 100% identical tothe sequence of a corresponding beta or beta-like strand of SEQ ID NO: 1or 6. Preferably, variations in the β-strand regions will not disruptthe stability of the polypeptide in physiological conditions. Inexemplary embodiments, the ¹⁰Fn3 domain binds to a desired target with aK_(d) of less than 500 nM, 100 nM, 50 nM, 1 nM, 500 pM, 100 pM or less.In some embodiments, the ¹Fn3 domain of a fibronectin based proteinscaffold binds to a desired target with a K_(d) between 1 pM and 1 μM,between 100 pM and 500 nM, between 1 nM and 500 nM, or between 1 nM and100 nM. In exemplary embodiments, the fibronectin based scaffold proteinbinds specifically to a target that is not bound by a wild-type ¹⁰Fn3domain, particularly the wild-type human ¹⁰Fn3 domain.

In some embodiments, the disclosure provides polypeptides comprising a¹⁰Fn3 domain, wherein the ¹Fn3 domain comprises a loop, AB; a loop, BC;a loop, CD; a loop, DE; a loop, EF; and a loop, FG; and has at least oneloop selected from loop AB, BC, CD, DE, EF and FG with an altered aminoacid sequence relative to the sequence of the corresponding loop of thehuman ¹⁰Fn3 domain of SEQ ID NO: 1. In some embodiments, the BC, DE andFG loops are altered. In other embodiments, the CD and FG loops arealtered. In other embodiments, the CD, DE and EF loops are altered. Inother embodiments, the EF and FG loops are altered. In otherembodiments, the AB, CD and EF loops are altered. In other embodiments,the FG loop is the only loop altered. In other embodiments, the CD andFG loops are both altered. In other embodiments, the CD and EF loops arealtered. In some embodiments, one or more specific scaffold alterationsare combined with one or more loop alterations. By “altered” is meantone or more amino acid sequence alterations relative to a templatesequence (i.e., the corresponding wild-type human fibronectin domain)and includes amino acid additions, deletions, and substitutions.

In some embodiments, the fibronectin based scaffold protein comprises a¹⁰Fn3 domain having an amino acid sequence at least 80, 85, 90, 95, 98,or 100% identical to the non-loop regions of SEQ ID NO: 1 or 6, whereinat least one loop selected from AB, BC, CD, DE, EF and FG is altered.For example, in certain embodiments, the AB loop may have up to 4 aminoacid substitutions, up to 10 amino acid insertions, up to 3 amino aciddeletions, or a combination thereof; the BC loop may have up to 10 aminoacid substitutions, up to 4 amino acid deletions, up to 10 amino acidinsertions, or a combination thereof; the CD loop may have up to 6 aminoacid substitutions, up to 10 amino acid insertions, up to 4 amino aciddeletions, or a combination thereof; the DE loop may have up to 6 aminoacid substitutions, up to 4 amino acid deletions, up to 13 amino acidinsertions, or a combination thereof, the EF loop may have up to 5 aminoacid substations, up to 10 amino acid insertions, up to 3 amino aciddeletions, or a combination thereof; and/or the FG loop may have up to12 amino acid substitutions, up to 11 amino acid deletions, up to 25amino acid insertions, or a combination thereof.

In certain embodiments, the fibronectin based scaffold protein comprisesa ¹⁰Fn3 domain that is defined generally by following the sequence:

(SEQ ID NO: 22) VSDVPRD LEVVAA (X)_(u) LLISW (X)_(v) YRITY (X)_(w) FTV(X)_(x) ATISGL (X)_(y) Y TITVYA (X)_(z) ISINY RT

In SEQ ID NO: 22, the AB loop is represented by (X)_(u), the BC loop isrepresented by (X)_(v), the CD loop is represented by (X)_(w), the DEloop is represented by (X)_(x), the EF loop is represented by (X), andthe FG loop is represented by X_(z). X represents any amino acid and thesubscript following the X represents an integer of the number of aminoacids. In particular, u, v, w, x, y and z may each independently beanywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15,6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. The sequences of the betastrands (underlined) may have anywhere from 0 to 10, from 0 to 8, from 0to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, deletions or additions across all 7 scaffold regionsrelative to the corresponding amino acids shown in SEQ ID NO: 22. Insome embodiments, the sequences of the beta strands may have anywherefrom 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0to 3, from 0 to 2, or from 0 to 1 conservative substitutions across all7 scaffold regions relative to the corresponding amino acids shown inSEQ ID NO: 22. In certain embodiments, the hydrophobic core amino acidresidues (bolded residues in SEQ ID NO: 22 above) are fixed, and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the hydrophobic core amino acid residues. In someembodiments, the hydrophobic core residues of the polypeptides providedherein have not been modified relative to the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1).

B. Scaffold Region Modifications

The non-loop sequences of ¹⁰Fn3, i.e., the “scaffold regions”, may bealtered provided that the ¹⁰Fn3 domain retains target binding functionand/or structural stability. In some embodiments, one or more of Asp 7,Glu 9, and Asp 23 are replaced by another amino acid, such as, forexample, a non-negatively charged amino acid residue (e.g., Asn, Lys,etc.). These mutations have been reported to have the effect ofpromoting greater stability of the mutant ¹⁰Fn3 at neutral pH ascompared to the wild-type form (See, PCT Publication No. WO 02/04523). Avariety of additional alterations in the ¹⁰Fn3 scaffold that are eitherbeneficial or neutral have been disclosed. See, for example, Batori etal., Protein Eng. 2002 15(12):1015-20; Koide et al., Biochemistry 200140(34):10326-33.

The scaffold regions, e.g., the β-strands and/or N- and C-termini, of¹⁰Fn3 may be modified to increase binding of the polypeptide to adesired target or to reduce immunogenicity. In some embodiments,residues involved in forming the hydrophobic core, i.e., residuescorresponding to residues L8, V10, A13, L18, I20, W22, Y32, I34, Y36,F48, V50, A57, I59, L62, Y68, I70, V72, A74, I88, I90 and Y92 of SEQ IDNO: 1 or 6 are not mutated. In some embodiments, any one of, or acombination of any one of, residues corresponding to residues 1-7, 9-15,19, 21, 33, 35, 36, 49, 58, 60, 61, 69, 71 73, 88, 89 and 91-101 of thescaffold region of the ¹⁰Fn3 polypeptide is mutated to a different aminoacid as compared to the corresponding amino acid present in the aminoacid sequence set forth as SEQ ID NO: 1 or 6.

In some embodiments, mutations may be made to the scaffold regions ofthe polypeptides, provided that the one or more of the followingspecific mutations are excluded: V1A; S2P; S2T; D3G; D3S; P5S; R6G; R6S;D7G; D7K; L8P; L8Q; E9D; E9K; E9R; E9V; V10A; V10I; A12D; A12E; A12V;L18E; L18I; L18P; L18Q; L18R; L19Q; S21C; S21G; S21N; R29G; R29S; R29Y;Y31H; Y32F; R33G; I34T; I34V; T35A; T35F; T35I; Y36H; F48L; F48S; T49A;T49I; V50A; V50E; V50M; A57Deletion; T58A; T58I; T58Deletion; I59VI59Deletion; S60G; S60N; S60R; G61C; G61R; L62R; D67G; D67K; D67N; Y68A;Y68D; T69I; I70N; I70S; I70V; T71A; V72A; V72G; Y73C; Y73H; A74G; A74T;I88S; I88T; I88V; S89P; 190F; 190T; 190V; N91D; N91S; N91T; Y92C; Y92H;Y92L; Y92R; Y92Deletion; R93Q; R93T and T94A. In certain embodiments,these specific scaffold mutations are excluded in the context of a ¹⁰Fn3domain in which the BC, DE and FG loops have been modified.

In some embodiments, mutations may be made to the scaffold regions ofthe polypeptides, provided that the following mutation combinations areexcluded:

-   -   L18R, S21C and S60G;    -   E9D, L18R, V50E and T56I;    -   L18R, T49I and N91D;    -   F48S and T71A;    -   P5S, V10A, S60G and S89P;    -   L18R, and Y92C;    -   L18R and F48S;    -   L18R and V72A;    -   L18Q, R33G and F48S;    -   Y68D and Y92H;    -   R6S, L62R and N91S;    -   L8P, E9V, I34V, T71A and Y92Deletion;    -   E9K, L18R and F48L;    -   E9R, L18R, S60G and I70V;    -   L18R, I88V and 190T;    -   L18R, N91D and Y92C;    -   L18R and I34T;    -   L18R and G61C;    -   Y32F, T71A and T94A;    -   L18R, T58A, Y92L and R93T;    -   V50M, T58A, S89P, 190F and Y92R;    -   S2T, D7G, E9K, V10I, T58A, S60N and S89P;    -   I59V, S60N and T94A;    -   R6G, S21G, T35A, T58I and S60G;    -   L18P, S21C, T58A, Y73H and Y92C;    -   Y31H, R33G and G61R;    -   A74G, R93Q and T94A    -   S2P and T58I;    -   T58I and I88T;    -   T58I and I90T;    -   G61R and A74T    -   A57Deletion, T58Deletion and I59Deletion    -   R33G, T35I and V50M    -   V1A, R33G and V50M    -   R33G and V50M    -   R33G, I34V and V50M    -   D3G, L181, R33G, V50M, Y73H and N91T    -   R6G, T35F and V72A    -   A12V, S21N and T35A    -   S21G and T49A    -   D3S and D7K    -   A12V and L19Q    -   A12D, L18I and L19Q    -   A12E, L18I and L19Q        In certain embodiments, these specific combinations of scaffold        mutations are excluded in the context of a ¹⁰Fn3 domain in which        the BC, DE and FG loops have been modified.

In some embodiments, polypeptides having mutations at a positioncorresponding to position 21 of SEQ ID NO: 1 or 6 are excluded, unless amutation at this position is combined with a mutation or mutations atany one of amino acid positions corresponding to positions 1-7, 19, 31,49, 58, 60, 73, 75 and 89 of SEQ ID NO: 1 or 6. In some embodiments,polypeptides having mutations at a position corresponding to position 60of SEQ ID NO: 1 are excluded unless a mutation at this position iscombined with a mutation or mutations at an amino acid positioncorresponding to any one of positions 1-7, 9-17, 19, 21, 23-31, 33, 35,49, 51-56, 65-67, 75-87 and 89 of SEQ ID NO: 1 or 6. In someembodiments, polypeptides having mutations at position 61 of SEQ ID NO:1 or 6 are excluded, unless a mutation at this position is combined witha mutation or mutations at an amino acid position corresponding to anyone of positions 11, 12, 19, 46, 66-67, 69 and 91 of SEQ ID NO: 1 or 6.In some embodiments, polypeptides having mutations at positions 93 or 94of SEQ ID NO: 1 or 6 are excluded, unless a mutation at either of thesepositions is combined with a mutation or mutations at a positioncorresponding to any one of amino acid positions 1-7, 9-14, 65-67, 89and 91 of SEQ ID NO: 1 or 6. In certain embodiments, these exclusionsapply in the context of a ¹⁰Fn3 domain in which the BC, DE and FG loopshave been modified.

In certain embodiments, the non-loop region of the ¹⁰Fn3 domain may bemodified by one or more conservative substitutions. As many as 5%, 10%,20% or even 30% or more of the amino acids in the ¹⁰Fn3 scaffold may bealtered by a conservative substitution without substantially alteringthe affinity of the ¹⁰Fn3 for a ligand. In certain embodiments, thescaffold may comprise anywhere from 0-15, 0-10, 0-8, 0-6, 0-5, 0-4, 0-3,1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4,5-15, or 5-10 conservative amino acid substitutions. In exemplaryembodiments, the scaffold modification preferably reduces the bindingaffinity of the ¹⁰Fn3 binder for a ligand by less than 100-fold,50-fold, 25-fold, 10-fold, 5-fold, or 2-fold. It may be that suchchanges will alter the immunogenicity of the ¹⁰Fn3 in vivo, and wherethe immunogenicity is decreased, such changes will be desirable. As usedherein, “conservative substitutions” are residues that are physically orfunctionally similar to the corresponding reference residues. That is, aconservative substitution and its reference residue have similar size,shape, electric charge, chemical properties including the ability toform covalent or hydrogen bonds, or the like. Preferred conservativesubstitutions are those fulfilling the criteria defined for an acceptedpoint mutation in Dayhoff et al., Atlas of Protein Sequence andStructure 5:345-352 (1978 & Supp.). Examples of conservativesubstitutions are substitutions within the following groups: (a) valine,glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d)aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine.

In some embodiments, a ¹⁰Fn3 molecule comprises the amino acid sequenceof any of the library designs set forth herein, e.g., in FIGS. 2, 9 and12 (or amino acids 1-94 thereof, SEQ ID NO: 6) and comprises at most 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 additional substitutions, additions ordeletions in a loop and/or a strand. In certain embodiments, a ¹⁰Fn3molecule comprises the amino acid sequence of any of the library designsset forth herein, e.g., in FIGS. 2, 9 and 12 (or amino acids 1-94thereof; SEQ ID NO: 6) with no other amino acid modifications. A ¹⁰Fn3molecule that comprises the amino acid sequence of any of the librarydesigns set forth herein, e.g., in FIGS. 2, 9 and 12 (or amino acids1-94 thereof; SEQ ID NO: 6) may comprise any amino acid at a variedposition, and in some instances even that of the wild-type ¹⁰Fn3molecule. In certain embodiments, a ¹⁰Fn3 molecule that comprises theamino acid sequence of any of the library designs set forth herein,e.g., in FIGS. 2, 9 and 12 (or amino acids 1-94 thereof; SEQ ID NO: 6)comprises only non wild-type amino acids at each of the positionsindicated as varied (those underlined or boxed, and bolded).

C. N- and C-Terminal Regions

In some embodiments, the amino acid sequences of the N-terminal and/orC-terminal regions of the polypeptides provided herein may be modifiedby deletion, substitution or insertion relative to the amino acidsequences of the corresponding regions of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 6). The ¹⁰Fn3 domains generally begin with aminoacid number 1 of SEQ ID NO: 1. However, domains with amino aciddeletions are also encompassed by the invention. In some embodiments,the first eight (i.e., residues 1-8) and the last seven amino acids(i.e., residues 95-101) of SEQ ID NO: 1 are deleted, generating a ¹⁰Fn3domain having the amino acid sequence of SEQ ID NO: 60. In certainembodiments, the last seven amino acids (i.e., residues 95-101) of SEQID NO: 1 are deleted, generating a ¹⁰Fn3 domain having the amino acidsequence of SEQ ID NO: 6. Additional sequences may also be added to theN- or C-terminus of a ¹⁰Fn3 domain having the amino acid sequence of SEQID NO: 1, 2, 6, or 60. For example, in some embodiments, the N-terminalextension consists of an amino acid sequence selected from the groupconsisting of: M, MG, and G.

In certain embodiments, the amino acid sequence of the first 1, 2, 3, 4,5, 6, 7, 8 or 9 residues of SEQ ID NO: 1 or 6 may be modified or deletedin the polypeptides provided herein relative to the sequence of thecorresponding amino acids in the wild-type human ¹⁰Fn3 domain (SEQ IDNO: 1 or 6). In exemplary embodiments, the amino acids corresponding toamino acids 1-8 of SEQ ID NO: 1 or 6 are replaced with an alternativeN-terminal region having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2,or 1 amino acids in length. Exemplary alternative N-terminal regionsinclude (represented by the single letter amino acid code) M, MG, G,MGVSDVPRDL (SEQ ID NO: 9) and GVSDVPRDL (SEQ ID NO: 11), or N-terminaltruncations of any one of SEQ ID NOs: 9 and 11. Other suitablealternative N-terminal regions include, for example, X_(n)SDVPRDL (SEQID NO: 16), XnDVPRDL (SEQ ID NO: 17), XnVPRDL (SEQ ID NO: 18), XnPRDL(SEQ ID NO: 19), XnRDL (SEQ ID NO: 20), XnDL (SEQ ID NO: 21), or XnL,wherein n=0, 1 or 2 amino acids, wherein when n=1, X is Met or Gly, andwhen n=2, X is Met-Gly. When a Met-Gly sequence is added to theN-terminus of a ¹⁰Fn3 domain, the M will usually be cleaved off, leavinga G at the N-terminus. In other embodiments, the alternative N-terminalregion comprises the amino acid sequence MASTSG (SEQ ID NO: 50).

In certain embodiments, the amino acid sequence corresponding to aminoacids 93-101, 94-101, 95-101, 96-101, 97-101, 98-101, 99-101, 100-101,or 101 of SEQ ID NO: 1 are deleted or modified in the polypeptidesprovided herein relative to the sequence of the corresponding aminoacids in the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). Inexemplary embodiments, the amino acids corresponding to amino acids95-101 of SEQ ID NO: 1 are replaced with an alternative C-terminalregion having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 aminoacids in length. Specific examples of alternative C-terminal regionsequences include, for example, polypeptides comprising, consistingessentially of, or consisting of, EIEK (SEQ ID NO: 7), EGSGC (SEQ ID NO:23), EIEKPCQ (SEQ ID NO: 24), EIEKPSQ (SEQ ID NO: 25), EIEKP (SEQ ID NO:26), EIEKPS (SEQ ID NO: 27), EIEKPC (SEQ ID NO: 8), or SEQ ID NO: 44. Insome embodiments, the alternative C-terminal region comprises EIDK (SEQID NO: 29), and in particular embodiments, the alternative C-terminalregion is either EIDKPCQ (SEQ ID NO: 31) or EIDKPSQ (SEQ ID NO: 30).

In certain embodiments, the fibronectin based scaffold proteins comprisea ¹⁰Fn3 domain having both an alternative N-terminal region sequence andan alternative C-terminal region sequence.

When referring herein to molecules comprising a particular librarydesign, which library design comprises amino acids 1-101 (SEQ ID NO: 1),it is understood that also encompassed herein are the same moleculescomprising amino acids 1-94 (SEQ ID NO: 6) and not including the 7N-terminal amino acids, and/or not including C-terminal amino acids.

D. Proteins Having Novel Loop and Scaffold Combinations

A “patch library”, as described herein, refers to a library in which aregion on the surface of the scaffold protein is diversified. Residuesto be diversified can be determined by picking one spot on the surfaceof the protein, then identifying all surface and loop residues withinsome distance (e.g., 8 Å), and adjusting for shape, sequenceconnectivity, conservation, etc. For example, to generate a patchlibrary centering on the “SouthWest” portion of the scaffold, Asp67, thelast amino acid in the EF loop, which is approximately centered on theSW side was selected. All residues within an 8 Å distance of Asp67 arethen identified, and the hydrophobic core residues are removed from thelist of residues to be diversified, providing a total of 14 amino acidsto randomize, including G37-G41, K63-D67, T69, and the C-terminalsequence beginning with N91 (residues positions numbered in accordancewith the sequence of the wild-type human ¹⁰Fn3 domain having SEQ ID NO:1 or 6). Further means of diversifications can then be incorporated intothe scaffold design, for example, varying the length of the CD loop (toallow greater shape variation) or modifying or extending the sequence ofthe N-terminal region. Examples of possible amino acid residues to bemutated in order to generate a representative patch library are providedin FIGS. 2A-F and 9A-C. Three-dimensional structures of ¹⁰Fn3 domainpeptides illustrating several of the different interfaces that may betargeted to generate a representative patch library are shown in FIGS.3A-F. In some embodiments, amino acids in a ¹⁰Fn3 domain polypeptide arediversified not with regard to loop definitions, but rather with regardto their physical location on the surface of the ¹⁰Fn3 structure. Insome embodiments, a ‘patch’ of 10 to 30 or more amino acids arediversified, chosen to form a generally contiguous surface that can spanboth loops and strands, or can be solely on strand residues.

1. Binders Having a North Pole and South Pole Loop Modified

In some embodiments, the polypeptides provided herein comprise a ¹⁰Fn3domain having (i) a modification in the amino acid sequence of at leastone north pole loop selected from the BC, DE and FG loops relative tothe corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1or 6), and (ii) a modification in the amino acid sequence of at leastone south pole loop selected from the AB, CD and EF loops relative tothe corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1or 6). The modified north pole and south pole loops contribute tobinding to the same target. Various combinations of modified north poleand south pole loops are contemplated. For example, a ¹⁰Fn3 may compriseone modified north pole loop and one modified south pole, one modifiednorth pole loop and two modified south pole loops, two modified northpole loops and one modified south pole loop, two modified north poleloops and two modified south pole loops, three modified north pole loopsand one modified south pool loop, etc., wherein each of the modifiedloops contributes to binding to the same target. Exemplary combinationsof north pole and south pole loops that may be modified include, forexample, the CD loop (south pole) and the FG loop (north pole), the CDloop (south pole) and the DE loop (north pole), the EF loop (south pole)and FG loop (north pole), the AB loop (south pole) and the FG loop(north pole), or the DE loop (north pole) and the EF loop (south pole).Another exemplary loop combination is the CD loop (south pole), the DEloop (north pole) and the EF loop (south pole). Yet another exemplaryloop combination is the DE loop (north pole) and one of more of the AB,CD and EF loops (south pole). The modified loops may have sequencemodifications across an entire loop or only in a portion of the loop.Additionally, one or more of the modified loops may have insertions ordeletions such that the length of the loop is varied relative to thelength of the corresponding loop of the wild-type sequence. In certainembodiments, additional regions in the ¹⁰Fn3 domain (i.e., in additionto the north and south pole loops), such as β-strand, N-terminal and/orC-terminal regions, may also be modified in sequence relative to thewild-type ¹⁰Fn3 domain, and such additional modifications may alsocontribute to binding to the target.

Exemplary ¹⁰Fn3 designs having at least one north pole loop and at leastone south pole loop modified include, for example, the WS1, WS2, WS3,WS2′, Front1, Front2, Back1, Back2, WS-LI1, SouthFront, and AG Stranddesigns shown in FIG. 9A and the West Side, South Front and AG Stranddesigns shown in FIGS. 2C-2E.

2. Binders Having Loop and Scaffold Region Modifications

Also provided herein are ¹⁰Fn3 domains having novel combinations of loopand scaffold modifications. In particular, the application providespolypeptides comprising a ¹⁰Fn3 domain comprising (i) a modification inthe amino acid sequence of at least one of loops AB, BC, CD, DE, EF, orFG, and (ii) a modification in the amino acid sequence of at least onescaffold region (i.e., a modification in at least one β-strand, theN-terminal region, and/or the C-terminal region), wherein the modifiedloop(s) and modified scaffold region(s) both contribute to binding thesame target. In exemplary embodiments, the scaffold region modificationsare located adjacent to modifications in a loop region, e.g., if the ABloop is modified, scaffold mutations may tend to be located in β-strandA and/or β-strand B, which are adjacent to the AB loop in the linearsequence of the ¹⁰Fn3 domain. In other embodiments, a cluster ofmodifications may be found together in loop and scaffold regions thatare adjacent to one another in the linear sequence of the ¹⁰Fn3 domain.For example, ¹⁰Fn3 binders having both loop and scaffold modifications,may have clusters of amino acid modifications in the followingcombinations of loop and scaffold regions that are adjacent to eachother in the linear sequence of the ¹⁰Fn3 domain:β-strand/loop/P3-strand, loop/β-strand/loop,loop/β-strand/loop/β-strand, terminal region/β-strand/loop, orloop/β-strand/terminal region, etc. For example, ¹⁰Fn3 domains havingnovel combinations of loop and scaffold modifications may have clustersof modifications such that over a stretch of 20 contiguous amino acidsat least 15 of the amino acids are modified relative to wild-type. Inother embodiments, at least 17 out of 20, 18 out of 20, 17 out of 25, 20out of 25, or 25 out of 30 residues in a contiguous stretch are modifiedrelative to the wild-type ¹⁰Fn3 domain sequence over the correspondingstretch of amino acids. In certain embodiments, a given ¹⁰Fn3 domain mayhave two or three clusters of modifications separated by stretches ofunmodified (i.e., wild-type) sequence. For any given region (i.e., aloop, β-strand or terminal region) that is modified, all or only aportion of the region may be modified relative to the wild-typesequence. When a β-strand region is modified, preferably the hydrophobiccore residues remain unmodified (i.e., wild-type) and one or more of thenon-core residues in the β-strand are modified. Suitable modificationsin the loop, β-strand or terminal regions include amino acidsubstitutions, deletion and/or insertions, as well as combinationsthereof.

Exemplary ¹⁰Fn3 designs having at least one loop region and at least onescaffold region modified include, for example, the WS1, Front1, Front2,Back1, Back2, SP1, SP2, SP3, SouthFront, AG Strand and SouthWest designsshown in FIG. 9A, the NW3 design shown in FIG. 9B, the NP1, NE1, NP4-5,NP6-1 and NW2 designs shown in FIG. 9C, and the designs shown in FIGS.2A-2F.

3. “West-Side” Binders

In some embodiments, the application provides ¹⁰Fn3 domains having abinding face along the “west-side” of the molecule (See FIG. 3C) and arereferred to as “West-side binders” or “WS binders”. WS binders asdescribed herein comprise a ¹⁰Fn3 domain that has a modified CD loop anda modified FG loop, as compared to the corresponding CD and FG loopsequences set forth in SEQ ID NO: 1 or 6. The CD loop and the FG loopboth contribute to binding to the same target. In certain embodiments,the WS binders may comprise additional modifications at one or moreregions within the ¹⁰Fn3 domain. For example, WS binders may comprisescaffold modifications in one or more of the β-strand regions adjacentto the CD and/or FG loops. In particular, WS binders may comprisesequence modifications in one or more of β-strand C, β-strand D,β-strand F, and/or β-strand G. Exemplary scaffold modifications includemodifications at one or more scaffold region positions corresponding tothe amino acid positions: 33, 35, 49, 69, 71, 73, 89 and/or 91 of SEQ IDNO: 1 or 6. The WS binders may also comprise modifications in the BCloop, particularly in the C-terminal portion of the BC loop. In oneembodiment, the last two residues of the BC loop (i.e., corresponding toamino acids 30 and 31 in the wild-type ¹⁰Fn3 domain) are modifiedrelative to the wild-type sequence. All or a portion of the additionalloop and scaffold modifications may contribute to binding to the targetin conjunction with the modified CD and FG loops. Preferably, thehydrophobic core residues are not modified relative to the wild-typesequence.

In certain embodiments, a WS binder has a CD loop that is about 3-11,4-9 or 5 residues long; an FG loop that is about 1-10, e.g., 6 or 5,residues long; a C strand that is about 6-14, 8-11, or 9 residues long;and/or an F strand that is about 9-11 or 10 residues long. Positions 31,33, 35 and 37-39 of the beta strand C may be altered relative to thewild-type sequence. Positions 32, 34 and 36 of the beta strand C may behydrophobic residues. Positions 67, 69, 71 and 73 of the beta strand Fmay be altered relative to the wild-type sequence. Positions 68, 70, and72 of the beta strand F may be hydrophobic residues. A WS binder maycomprise amino acid substitutions at positions 30, 31, 32, 33, 34, 35,36, 37, 38 and/or 39, such as positions 31, 33, 35, 37, 38 and/or 39,e.g., positions 31 and/or 33, of SEQ ID NO: 1 or 6. A WS binder maycomprise amino acid substitutions at positions 44, 45, 46, 47, 48, 49,50 and/or 51, such as positions 44, 45, 47 and/or 49, of SEQ ID NO: 1 or6. A WS binder may comprise amino acid substitutions at positions 40,41, 42, 43, 44 and/or 45 of SEQ ID NO: 1 or 6. A WS binder may compriseamino acid substitutions at positions 67, 68, 69, 70, 71, 72, 73, 74, 75and/or 76, such as positions 67, 69, 71, 73 and/or 76 or positions 71,73, 75 and/or 76, of SEQ ID NO: 1 or 6. A WS binder may comprise aminoacid substitutions at positions 76, 77, 78, 79, 81, 82, 83, 84, 85and/or 86, such as positions 84 and/or 85 of SEQ ID NO: 1 or 6. A WSbinder may comprise amino acid substitutions at positions 85, 86, 87,88, 89, 90, 91, 92, 93 and/or 94 of SEQ ID NO: 1 or 6. A WS binder maycomprise amino acid substitutions at positions 31, 33, 47, 49, 73 and/or75 of SEQ ID NO: 1 or 6. A WS binder may comprise a loop C comprisingfrom 4-9 varied, e.g., non wild-type amino acids; an FG loop comprisingfrom 5-6 varied, e.g., non wild-type amino acids; and wherein aminoacids 31, 33, 35, 37-39, 67, 69, 71, 73 and 76 are not wild-type. “Notwild-type” amino acids are amino acids that are not those found at thesame position in the wild-type human ¹⁰Fn3 molecule (having, e.g., SEQID NO: 1 or 6).

Exemplary ¹⁰Fn3 WS binder designs include, for example, the WS1, WS2,WS3, WS2′, and WS-LI1 designs shown in FIG. 9A and the design shown inFIG. 2C (or amino acids 1-94 thereof). When referring to a ¹⁰Fn3molecule having a particular design based on a ¹⁰Fn3 sequence comprisingamino acids 1-101, the description is intended to encompass thosemolecules that do not comprise “DK” at the end terminus and/or that donot comprise the N-terminal 7 amino acids, and correspond to amino acids1-94 of the sequence shown. Alternatively, when the design showncomprises amino acids 1-94, the description is intended to encompass thesame design with the N-terminal 7 amino acids, which may devoid of thesequence “DK.” Other modifications that may be made are describedherein.

Exemplary WS binder designs are provided in FIG. 12. WS3, for examplecorresponds to WS1, wherein the length of the loops CD and FG may bemodified, and D67 may also be modified. An example of a molecule havinga WS3 design is PXR binder having SEQ ID NO: 49. Variants of WS1, WS2,WS3, WS2′, WS-LI1, and WS4 include those having a wild-type or mutatedamino acid at positions 30, 31, 33, 35, 37, 38, 46, 47, 49, 50, 67, 69,71, 73, 75, 76, 84, 85, 86, 87, 89 or 91. For example, a WS binderdesign may comprise one or more amino acid modifications in amino acids39-45 of the CD loop and one or more amino acid modification in aminoacids 77-83 in loop FG (WS-LI1 design), and wherein a ¹⁰Fn3 moleculehaving that design binds specifically to a target molecule (andoptionally does not comprise an RGD sequence). A WS binder design maycomprise the design of WS-LI1 and at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 15, 17, 20 or 25 additional amino acid modifications in the loops orstrands. For example, a WS binder design may comprise the design ofWS-LI1 and at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or 25additional amino acid modifications at amino acid positions such as atamino acid positions 37, 38, 46, 47, 75, 76, and 85-88. Other amino acidmodifications that may be included are those at positions 30, 31, 33,35, 49, 50, 67, 69, 71, 73, 89 and 91. An exemplary WS design maycomprise the amino acid sequence of WS7 (FIG. 12), wherein the loops mayvary in length from those in the wild-type ¹⁰Fn3 molecule, and whereineach varied position (bolded and underlined) may be modified to anyother amino acid, or in certain instances, may be kept unmodified,provided that a ¹⁰Fn3 molecule with such a design binds specifically toa target molecule (and optionally does not comprise an RGD site). Incertain embodiments, a ¹⁰Fn3 molecule comprises the amino acid sequenceof WS7, wherein the length of loops CD and FG may be varied, wherein noother amino acid may be varied, and wherein exactly or at most 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or 25 amino acid residues thatare indicated as variable (those underlined and bolded) are actually notchanged, and are the amino acids corresponding to those at the sameposition in the wild-type human ¹⁰Fn3 molecule, i.e., wild-type aminoacids (SEQ ID NO: 1 or 6). For example, one or more of amino acids 30,31, 33, 35, 36, 37, 47, 49, 50, 67, 69, 71, 73, 75 and 87 in WS7 may bethe wild-type amino acid, provided that the WS binder binds specificallyto its target. In certain embodiments, a WS binder having a WS7 designdoes not comprise any amino acid modification other than thoseindicated. In certain embodiments, a WS binder having a WS7 designcomprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or 25amino acid modifications in addition to those indicated.

Also provided are libraries comprising any one of the WS binder designsdescribed herein. An exemplary library is a library comprising WSbinders having a varied CD and FG loop and further comprising nonwild-type amino acids at positions 30, 31, 33, 47 and 49. An exemplarylibrary is a library having a varied FG loop and further comprising nonwild-type amino acids at positions 30, 31, 33, 47 and 49.

In certain embodiments, at least or at most 10, 20, 30, 40, 50, or 60amino acids of a design sequence is not varied, e.g., is not varied bysubstitution. For example, one or more of the following amino acids areretained as the amino acid from the wild-type human ¹⁰Fn3 molecule:amino acids at positions 1-29, 32, 34, 36, 48, 51-66, 68, 70, 72, 88, 90and 92-101.

Examples of WS binders that bind specifically to therapeutic targets aredescribed in the Examples, and include for example polypeptides havingthe amino acid sequence of any one of SEQ ID NOs: 3-5, 45-49, 62-63, 66,and 72.

In some embodiments, a WS binder comprises the amino acid sequence ofWS1, WS2, WS3, WS2′, WS-LI1, WS4, WS5, WS6 or WS7 (or amino acids 1-94thereof) and comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10additional substitutions, additions or deletions in a loop and/or astrand. In certain embodiments, a WS binder comprises the amino acidsequence of WS1, WS2, WS3, WS2′, WS-LI1, WS4, WS5, WS6 or WS7 (or aminoacids 1-94 thereof) with no other amino acid modifications. A WS binderthat comprises the amino acid sequence of WS1, WS2, WS3, WS2′, WS-LI1,WS4, WS5, WS6 or WS7 (or amino acids 1-94 thereof) may comprise anyamino acid at a varied position, and in some instances even that of thewild-type ¹⁰Fn3 molecule. In certain embodiments, a WS binder thatcomprises the amino acid sequence of WS1, WS2, WS3, WS2′, WS-LI1, WS4,WS5, WS6 or WS7 (or amino acids 1-94 thereof) comprises non wild-typeamino acids at each of the positions indicated as varied (thoseunderlined and bolded).

4. “Front” Binders

In some embodiments, the polypeptides provided herein comprise a ¹⁰Fn3domain having modifications in the CD, DE and, in some cases, EF loops,wherein the loop modifications all contribute to target binding. Thesepolypeptides are referred to as “front binders” herein. The frontbinders may additionally comprise modifications in one or more scaffoldregions, particularly in scaffold regions that flank or are adjacent toa modified loop region. For example, the front binders may comprise ascaffold modification in one or more of β-strand C, β-strand D, and/orβ-strand E relative to the sequences of the corresponding β-strands ofthe wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). Preferably thehydrophobic core residues are not modified relative to the wild-typesequence. Exemplary scaffold modifications that may be present in frontbinders, include modifications at one or more positions corresponding toamino acid positions 36, 49, 58 and/or 60 of SEQ ID NO: 1 or 6. Suchscaffold modifications may contribute to binding to the target togetherwith the modified loops. In certain embodiments, the front binders maycomprise clusters of modifications spanning several loop and strandregions of the ¹⁰Fn3 domain. In particular, the front binders maycomprise modifications in at least 15, 20, 24, 25, or 27 of the 31residues between the amino acids corresponding to residues 36 through 66of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6). The loop and/orstrand modifications may include amino acid substitutions, deletionsand/or insertions, or combinations thereof. In exemplary embodiments,the CD loop is extended in length or reduced in length relative to theCD loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1 or 6).Exemplary ¹⁰Fn3 front binder designs include, for example, the Front1and Front2 designs shown in FIG. 9A.

5. “Back” Binders

In some embodiments, the polypeptides provided herein comprise a ¹⁰Fn3domain having modifications in the EF and FG loops, wherein the loopmodifications contribute to binding the same target. These polypeptidesare referred to as “back binders” herein. The back binders may compriseadditional modifications in other loop and/or scaffold regions. Forexample, a back binder may contain modifications in at least a portionof the AB loop, preferably the N-terminal portion of the AB loop. In anexemplary embodiment, the first two amino acids of the AB loop (i.e.,corresponding to amino acid residues 14 and 15 of the wild-type ¹⁰Fn3domain) are modified relative to the wild-type sequence. In certainembodiments, a back binder may also contain one or more scaffoldmodifications, particularly modifications in one or more scaffoldregions that are adjacent to a modified loop region. For example, backbinders may contain one or more modifications in one or more of β-strandA, β-strand G, the N-terminal region, and/or the C-terminal region.Preferably the hydrophobic core residues are not modified relative tothe wild-type sequence. Exemplary scaffold modifications includemodifications at one or more positions corresponding to amino acidpositions 1-7, 9-13, 89, 91, 93 and/or 94 of SEQ ID NO: 1 or 6. One ormore of the additional loop and/or scaffold modifications may contributeto binding to the target along with the modified EF and FG loops.Suitable loop and/or scaffold region modifications include amino acidsubstitutions, deletions and/or insertions, or combinations thereof. Incertain embodiments, the amino acid sequence of the FG loop is extendedin length or reduced in length relative to the FG loop of the wild-typehuman ¹⁰Fn3 domain (SEQ ID NO: 1 or 6).

In certain embodiments, a back binder may comprise a cluster of modifiedamino acid residues over a contiguous span of several regions in the¹⁰Fn3 domain. For example, at least 14 of the first 15 amino acidresidues of the ¹⁰Fn3 domain may be modified relative to thecorresponding residues in the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1or 6), and/or at least 15 of the 18 residues between the amino acidscorresponding to residues 80 through 97 (or 94) of the wild-type human¹⁰Fn3 domain (SEQ ID NO: 1 or 6) may be modified relative to thecorresponding residues in the wild-type sequence.

Exemplary ¹⁰Fn3 back binder designs include, for example, the Back1 andBack2 designs shown in FIG. 9A.

6. “South Pole” Binders

In certain embodiments, the application provides polypeptides comprisinga ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain comprises modifications in theamino acid sequences of β-strand A, loop AB, β-strand B, loop CD,β-strand E, loop EF, and β-strand F, relative to the sequences of thecorresponding regions of the wild-type sequence. These polypeptides arereferred to as “south pole binders” or “SP binders” herein. The modifiedloops and strands contribute to binding to the same target. The aminoacid sequence of the CD loop may be extended in length or reduced inlength relative to the CD loop of the wild-type human ¹⁰Fn3 domain (SEQID NO: 1 or 6). The south pole binders may comprise additionalmodifications in β-strand G and/or the C-terminal region relative to thesequence of the corresponding region of the wild-type sequence. Inexemplary embodiments, the south pole binders may comprise one or moremodifications at amino acids corresponding to positions 11, 12, 19, 60,61, 69, 91, 93 and 95-97 of the wild-type sequence. Exemplary ¹⁰Fn3south pole binder designs include, for example, the SP1, SP2 and SP3designs shown in FIG. 9A.

7. “Northwest” Binders

In some embodiments, the application provides polypeptides comprising a¹⁰Fn3 domain having modified BC, DE and FG loops, as compared to thecorresponding BC, DE and FG loop sequences set forth in SEQ ID NO: 1 or6, as well as additional modifications in one or more of β-strand C,β-strand D, β-strand F and β-strand G strand residues. The β-strand andloop region modifications together contribute to binding to the target.These proteins are referred to as “Northwest binders”, or “NW binders”,herein. In exemplary embodiments, the NW binders comprise one or morescaffold modifications at any one of, or combination of, amino acidpositions corresponding to scaffold region positions R33, T49, Y73 andS89 of SEQ ID NO: 1 or 6. Suitable modifications in loop and scaffoldregions include amino acid substations, deletions and/or insertions, orcombinations thereof. In certain embodiments, one or more of the BC, DEand FG loops are extended in length or reduced in length, orcombinations thereof, relative to the wild-type sequence. In oneembodiment, each of the BC, DE and FG loops are extended in length orreduced in length, or combinations thereof, relative to the wild-typesequence (e.g., SEQ ID NO: 1 or 6). In certain embodiments, only aportion of the BC loop is modified, particularly the C-terminal portion,relative to the wild-type sequence. For example, the BC loop may bemodified only at amino acid residues corresponding to amino acids 27-31of the wild-type BC loop, whereas the rest of the BC loop (i.e.,corresponding to residues 23-26 of the wild-type loop) are leftunmodified.

Exemplary ¹⁰Fn3 NW binder designs include, for example, the NW3 designshown in FIG. 9B, the NW2 design shown in FIG. 9C, and the design shownin FIG. 2A. A model of the NW binder is depicted in FIG. 3A.

8. “Northeast” Binders

In some embodiments, the application provides polypeptides comprising a¹⁰Fn3 domain having a modified BC, DE and FG loop as well as one or moreadditional modifications in any one of, or combination of, theN-terminal region, β-strand A, β-strand B and/or β-strand E. Theseproteins are referred to as “Northeast binders”, or “NE binders”,herein. In exemplary embodiments, the NE binders are modified at any oneof, or combination of, amino acids corresponding to scaffold regionpositions 1-7, E9, L19, S21 and/or T58 of the wild-type sequence (SEQ IDNO: 1 or 6). The combination of modified loop and scaffold regionscontributes to binding to the target. Exemplary ¹⁰Fn3 NE binder designsinclude, for example, the NE1 design shown in FIG. 9C and the designshown in FIG. 2B. A model of the NE binder is depicted in FIG. 3B.

9. “South Front” Binders

In some embodiments, the application provides polypeptides comprising a¹⁰Fn3 domain having modifications in one or more of the AB, CD, DE andEF loops, as well as additional modifications in one or more of β-strandB, β-strand D and/or β-strand E. These proteins are referred to as“South Front binders” herein. The combination of modified loop andstrand residues contributes to binding to the target. In exemplaryembodiments, a South Front binder may be modified at one or more aminoacid positions corresponding to scaffold region positions L19, T49, T58,S60, and/or G61 of SEQ ID NO: 1 or 6 and/or at one or more amino acidpositions corresponding to loop region positions T14-S17, P51, T56,G40-E47, and/or K63-G65 of SEQ ID NO: 1 or 6. In exemplary embodiments,a South Front binder may be extended in length or reduced in length inthe AB loop, between amino acids corresponding to residues 18 and 20 ofthe wild-type sequence, and/or in the CD loop. Exemplary ¹⁰Fn3 SouthFront binder designs include, for example, the SouthFront design shownin FIG. 9A and the design shown in FIG. 2D. A model of the South Frontbinder is depicted in FIG. 3D.

10. “AG” Binders

In some embodiments, the application provides polypeptides comprising a¹⁰Fn3 domain having a modified β-strand A and β-strand G, as compared tothe corresponding strand of SEQ ID NO: 1 or 6. These proteins arereferred to as “AG Binders” or “AG Strand” binders herein. In certainembodiments, the AG strand binders comprise clusters of modifications atthe N-terminal and C-terminal portions of the ¹⁰Fn3 domain, whereas themiddle portion of the ¹Fn3 remains unmodified. For example, an AG strandbinder may comprise modifications at 16 out of 19 of the first 19 aminoacids in the ¹⁰Fn3 domain (i.e., corresponding to amino acid positions1-19 of SEQ ID NO: 1 or 6) and modifications at 13-17 out of 18 of thelast 18 amino acids in the ¹⁰Fn3 domain (i.e., corresponding to aminoacid positions 84-101 of SEQ ID NO: 1) or at 14-18 out of 22 of the last22 amino acids in the ¹⁰Fn3 domain (i.e., corresponding to amino acidpositions 80-101 of SEQ ID NO: 1). In exemplary embodiments, an AGbinder may comprise modifications at one or more positions correspondingto positions 1-7, 9, 11-17, 19, 84-89 and 91-97 of SEQ ID NO: 1.Preferably the modified regions in an AG binder contribute to binding tothe same target. Exemplary ¹⁰Fn3 AG binder designs include, for example,the AG Strand design shown in FIG. 9A and the design shown in FIG. 2E. Amodel of the AG binder is depicted in FIG. 3E.

11. “Southwest” Binders

In some embodiments, the application provides polypeptides comprising a¹⁰Fn3 domain having a modified CD and EF loop, as well as additionalmodifications in any one of, or combination of residues corresponding topositions 69 or 91-97 of SEQ ID NO: 1. These proteins are referred to as“Southwest binders”, or “SW binders”, herein. The modified loop andscaffold regions contribute to binding to the target. Exemplary ¹⁰Fn3 SWbinder designs include, for example, the SouthWest design shown in FIG.9A and the design shown in FIG. 2F. A model of the SW binder is depictedin FIG. 3F.

E. Proteins Having Reduced Immunogenicity

In some embodiments, the polypeptides provided herein are associatedwith reduced immunogenicity. As described in the examples, the regionaround the BC loop of a ¹⁰Fn3 domain appears to be an immunogenic hotspot. Accordingly, the application provides two types of ¹⁰Fn3 designshaving reduced immunogenicity. In the first type of design, the BC loopis left entirely or at least partially unmodified such that the host(e.g., human) immune response is more likely to recognize the BC regionof the ¹⁰Fn3 domain as self thereby avoiding an immune response. In thesecond type of design, the strong HLA binding anchor in the BC region ofthe ¹⁰Fn3 domain is removed or destroyed such that the BC region shouldnot bind as tightly to the host HLA receptors thereby decreasing theimmunogenic potential of the BC region of the ¹⁰Fn3 binders. These ¹⁰Fn3designs are described further below.

In certain embodiments, the application provides polypeptides havingreduced immunogenicity comprising a 10Fn3 domain wherein the entire BCloop is left as wild-type. Preferably such polypeptides have lowerimmunogenicity relative to an equivalent polypeptide with modificationsin the BC loop. Polypeptides with wild-type BC loops have modificationsin other regions of the ¹⁰Fn3 domain that are involved in targetbinding. Preferably, the modifications outside of the BC loop do notlead to a strong immune response to the ¹⁰Fn3 domain in the host.Examples of ¹⁰Fn3 binders where the entire BC loop is left as wild-typeinclude, for example, the WS binders, Front binders, Back binders, SouthPole binders, South Front binders, AG binders and Southwest binders asdescribed herein. Particular examples of ¹⁰Fn3 designs having the BCloop unmodified relative to the wild-type sequence, include, forexample, the WS2, WS3, Front1, Front2, Back1, Back2, SP1, SP2, SP3,LI-3(a), WS-LI1, LU-S9, SouthFront, AG Strand and SouthWest designsshown in FIG. 9A and the designs shown in FIGS. 2D-2F. In ¹⁰Fn3 binderdesigns having a wild-type BC loop, it may be desirable to leave all ora portion of β-strand B and/or β-strand C unmodified relative to thewild-type sequence as well, particularly the portions of β-strand Band/or β-strand C that are adjacent to the BC loop (i.e., the C-terminalportion of β-strand B and/or the N-terminal portion of β-strand C). Inexemplary embodiments, ¹⁰Fn3 domains having a wild-type BC loop andreduced immunogenicity may not have any modifications in the portion ofthe ¹⁰Fn3 domain that is N-terminal to the CD loop, i.e., the N-terminalregion, β-strand A, AB loop, β-strand B, BC loop and β-strand C are allleft unmodified relative to the wild-type sequence.

In certain embodiments, the application provides polypeptides havingreduced immunogenicity comprising a 10Fn3 domains wherein a portion ofthe BC loop is left as wild-type. Preferably such polypeptides havelower immunogenicity relative to an equivalent polypeptide withmodifications in a greater portion of the BC loop. In exemplaryembodiments, the N-terminal portion of the BC loop is left as wild-type.For example, the first 1, 2, 3, 4, 5, or 6 residues of the BC loop maybe left as wild-type, while the remaining C-terminal residues of the BCloop can be modified. In ¹⁰Fn3 designs having at least a portion of theN-terminal region of the BC loop as wild-type, it may be desirable toleave all or a portion of β-strand B and/or β-strand C unmodifiedrelative to the wild-type sequence as well, particularly the portions ofβ-strand B and/or β-strand C that are adjacent to the BC loop (i.e., theC-terminal portion of β-strand B and/or the N-terminal portion ofβ-strand C). In exemplary embodiments, ¹⁰Fn3 domains having thewild-type sequence in an N-terminal portion of the BC loop and reducedimmunogenicity may not have any modifications in the N-terminal region,β-strand A, AB loop, and β-strand B. In ¹⁰Fn3 designs with a portion ofthe BC loop as wild-type, the modified portion of the BC loop maycontribute to target binding along with modifications in other regionsof the ¹⁰Fn3 domain. Examples of ¹⁰Fn3 binders where an N-terminalportion of the BC loop is left as wild-type include, for example, the¹⁰Fn3 designs shown in FIG. 9B, the WS1 design shown in FIG. 9A, and the¹⁰Fn3 design shown in FIG. 2C.

In certain embodiments, the application provides polypeptides havingreduced immunogenicity comprising ¹⁰Fn3 domains, wherein the strong HLAanchor in the region of β-strand B/BC loop/β-strand C (the “BC anchor”)has been removed or destroyed (e.g., modified relative to the wild-typesequence in a manner that reduces binding affinity to one or more HLAreceptors). For example, the BC anchor may be removed or destroyed bymodifying the ¹⁰Fn3 domain at one or more positions corresponding topositions L19, S21, R33 and/or T35 of SEQ ID NO: 1 or 6. When the BCanchor has been removed or destroyed, it is possible to modify thesequence of the BC loop without significantly increasing the immunogenicpotential of the BC region. Accordingly, many such ¹⁰Fn3 designs havemodifications in the BC loop in addition to the modifications inβ-strand B and/or β-strand C. The BC loop may contribute to targetbinding, optionally in combination with modifications in other regionsof the ¹⁰Fn3 domain. The modifications in β-strand B and/or β-strand Cmay or may not contribute to target binding. Examples of ¹⁰Fn3 binderswhere the BC anchor has been removed or destroyed include, for example,the ¹⁰Fn3 designs shown in FIG. 9C and the ¹⁰Fn3 design shown in FIG.2B.

In certain embodiments, the polypeptides described herein have reducedimmunogenicity as compared to a polypeptide having SEQ ID NO: 61, e.g.,the immunogenicity of the polypeptide is lower than the immunogenicityof a polypeptide having SEQ ID NO: 61.

The immunogenicity of a polypeptide described herein may be assessed,for example, by one or more of the following methods: Human LeukocyteAntigen (“HLA”) binding, in silico prediction of HLA binding (forexample, with the Epimatrix program), in vitro activation of humanT-cells, in vivo animal immune response, or other methods for evaluatingimmunogenicity potential.

In certain embodiments, immunogenicity may be assessed by HLA bindingexperiments. Preferably the polypeptides provided herein bind to one ormore HLA receptors with an IC₅₀ that is less than or equal to the IC₅₀associated with binding between an equivalent HLA receptor(s) and awild-type ¹⁰Fn3 domain. For example, the polypeptides provided hereinmay bind to an HLA receptor with an IC₅₀ of greater than 10 μM, 15 μM,20 μM, 25 μM, 50 μM, 100 μM, 150 μM or 200 μM. In some embodiments, thepolypeptides may bind to an HLA receptor with an IC₅₀ between 10 μM and1 mM, between 100 μM and 1 mM or between 500 μM and 1 mM. The HLA alleleused for assessing the polypeptide/HLA IC₅₀ binding may be one or moreof DRB*0101, DRB*0301, DRB*0401, DRB*0701 and/or DRB*1501.

In some embodiments, immunogenicity may be assessed by in silicoanalysis, such as EpiMatrix. In particular embodiments, the polypeptidesprovided herein are associated with an EpiMatrix “Z” scale score lessthan or equal to the score associated with a wild-type ¹⁰Fn3 domain. Incertain embodiments, the polypeptides provided herein are associatedwith an EpiMatrix “Z” scale score that is no greater than 200% of thescore associated with a wild-type ¹⁰Fn3 domain. In some embodiments, thepolypeptides are associated with an EpiMatrix score less than 1.64 onthe EpiMatrix “Z” scale (An Z; 2009; Therapeutic Monoclonal Antibodies:From Bench to Clinic; John Wiley and Sons; New Jersey; pages 428-429).

In some embodiments, immunogenicity may be assessed by in vivo animalimmune response experiments. For example, an animal, e.g. mouse ormonkey, may be injected with the polypeptides provided herein and theIgG and/or IgM immune response measured. Preferably, the polypeptidesdescribed herein display an IgG or IgM immune response that is no morethan 200%, 100%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2% or 1% greaterthan the IgG or IgM immune response observed in a mouse or monkeyinjected with a wild-type ¹⁰Fn3 domain.

The application also provides libraries of the polypeptides providedherein and methods of selecting binders to a desired target from thelibraries. There will be a higher likelihood of isolating a targetbinding molecule with acceptable immunogenicity characteristics from thelibraries provided herein, relative to libraries that have not beendesigned to avoid the immunogenic potential associated with the BCregion. These libraries are useful for reducing the amount of effortnecessary to deimmunize polypeptide candidates, and to increase theprobability of identifying non-immunogenic polypeptide molecules.

F. North Pole Variants

Additional ¹⁰Fn3 designs disclosed herein include those havingvariations (or variable regions) in the N-terminus; BC loop and/or aminoacids around the BC loop; DE loop and/or amino acids around the DC loop;and/or FG loop and/or amino acids around the FG loop. Exemplary ¹⁰Fn3designs are set forth in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or6 are represented by x₁₋₉Lx, wherein x is either any amino acid or x isany amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the variable (i.e., x)positions. In certain embodiments, the amino acid sequence x₁₋₉Lx is notidentical to the amino acid sequence corresponding to amino acids 1-9 ofSEQ ID NO: 1. Exemplary ¹⁰Fn3 designs having amino acids represented byx₁₋₉Lx include, e.g., LI-39(a)-(l), LI-39B(a)-(j), LI-39F(a)-(h), andLI-59(a)-(f), which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 1-9 of SEQ ID NO: 1are represented by x₁₋₉Lx, linked (e.g., covalently linked) at itsN-terminus to GVSDVPGGSG or 1, 2, 3, 4, 5, 6, 7, 8, or 9 consecutiveamino acids thereof. Exemplary N-terminal amino acid sequences of ¹⁰Fn3domains may comprise: Gx₁₋₉Lx, SGx₁₋₉Lx, GSGx₁₋₉Lx, GGSGx₁₋₉Lx,PGGSGx₁₋₉Lx, VPGGSGx1.9Lx, DVPGGSGx₁₋₉Lx, SDVPGGSGx₁₋₉Lx,VSDVPGGSGx₁₋₉Lx, GVSDVPGGSGx₁₋₉Lx, GVx₁₋₉Lx, GVSx₁₋₉Lx, GVSDx₁₋₉Lx,GVSDVx₁₋₉Lx, GVSDVPx₁₋₉Lx, GVSDVPGx₁₋₉Lx, GVSDVPGGx₁₋₉Lx,GVSDVPGGSx₁₋₉Lx, wherein x is either any amino acid or x is any aminoacid except the amino acid that occurs at the corresponding position inSEQ ID NO: 1. These sequences may be linked to V10 of a ¹⁰Fn3 domain. Incertain embodiments, the amino acid sequence x₁₋₉Lx is not identical tothe amino acid sequence corresponding to amino acids 1-9 of SEQ IDNO: 1. Exemplary ¹⁰Fn3 designs having amino acids represented by x₁₋₉Lxinclude, e.g., LI-39(a)-(f), LI-39B(a)-(e), LI-39F(a)-(d), andLI-59(a)-(c), which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 28-29 of SEQ ID NO: 1are represented by xx, wherein x is either any amino acid or x is anyamino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1. In certain embodiments, the amino acidsequence xx is not identical to the amino acid sequence corresponding toamino acids 28-29 of SEQ ID NO: 1. Exemplary ¹⁰Fn3 designs having aminoacids 28-29 represented by xx include, e.g., LI-3(b), which is shown inFIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 23-29 of SEQ ID NO: 1are represented by x₇, wherein x is either any amino acid or x is anyamino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1. In certain embodiments, x is not the aminoacid that occurs at the corresponding position in SEQ ID NO: 1 or 6 in1, 2, 3, 4, 5, 6 or 7 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₇ is not identical to the aminoacid sequence corresponding to amino acids 23-29 of SEQ ID NO: 1.Exemplary ¹⁰Fn3 designs having amino acids 23-29 represented by x₇include, e.g., LI-5, LI-51, LI-52, LI-53 and LI-54, which are shown inFIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 25-29 of SEQ ID NO: 1are represented by x₅, wherein x is either any amino acid or x is anyamino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4 or 5 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₅ is not identical to the aminoacid sequence corresponding to amino acids 25-29 of SEQ ID NO: 1.Exemplary ¹⁰Fn3 designs having amino acids 25-29 represented by x₅include, e.g., LI-31(a), LI-32(a), LI-59(a) and LI-59(d), which areshown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 26-29 of SEQ ID NO: 1are represented by x₄, wherein x is either any amino acid or x is anyamino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3 or 4 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₄ is not identical to the aminoacid sequence corresponding to amino acids 26-29 of SEQ ID NO: 1.Exemplary ¹⁰Fn3 designs having amino acids 26-29 represented by x₄include, e.g., LI-31(b), LI-32(b), LI-59(b) and LI-59(e), which areshown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 27-29 of SEQ ID NO: 1are represented by x₃, wherein x is either any amino acid or x is anyamino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2 or 3 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₃ is not identical to the aminoacid sequence corresponding to amino acids 27-29 of SEQ ID NO: 1.Exemplary ¹⁰Fn3 designs having amino acids 27-29 represented by x₃include, e.g., LI-31(c), LI-32(c), LI-59(c) and LI-59(f), which areshown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 24-28 of SEQ ID NO: 1or 6 are represented by x₅, wherein x is either any amino acid or x isany amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, the amino acidsequence x₅ is not identical to the amino acid sequence corresponding toamino acids 24-28 of SEQ ID NO: 1. Exemplary ¹⁰Fn3 designs having aminoacids 24-28 represented by x₅ include, e.g., LI-38(a), LI-38(d),LI-39(a), LI-39(d), LI-39(g), LI-39(j), LI-39B(a) and LI-39B(f), whichare represented in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 25-28 of SEQ ID NO: 1or 6 are represented by x₄, wherein x is either any amino acid or x isany amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3 or 4 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₄ is not identical to the aminoacid sequence corresponding to amino acids 25-28 of SEQ ID NO: 1.Exemplary ¹⁰Fn3 designs having amino acids 25-28 represented by x₄include, e.g., LI-38(b), LI-38(e), LI-39(b), LI-39(e), LI-39(h),LI-39(k), LI-39B(b) and LI-39B(g), which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 26-28 of SEQ ID NO: 1or 6 are represented by x₃, wherein x is either any amino acid or x isany amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2 or 3 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₃ is not identical to the aminoacid sequence corresponding to amino acids 26-28 of SEQ ID NO: 1 or 6.Exemplary ¹⁰Fn3 designs having amino acids 26-28 represented by x₃include, e.g., LI-38(c), LI-38(f), LI-39(c), LI-39(f), LI-39(i),LI-39(l), LI-39B(c) and LI-39B(h), which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 17-26 of SEQ ID NO: 1or 6 are represented by xLxIxWx₄, wherein x is either any amino acid orx is any amino acid except the amino acid that occurs at thecorresponding position in SEQ ID NO: 1 or 6. In certain embodiments, xis not the amino acid that occurs at the corresponding position in SEQID NO: 1 or 6 in 1, 2, 3, 4, 5, 6 or 7 of the variable (i.e., x)positions. In certain embodiments, the amino acid sequence xLxIxWx₄ isnot identical to the amino acid sequence corresponding to amino acids17-26 of SEQ ID NO: 1. Exemplary ¹⁰Fn3 designs having amino acids 17-26represented by x₄ include, e.g., LI39B(e), LI-39B(j), NP4E1, NP4E2 andNP4E3, which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 24-35 of SEQ ID NO: 1or 6 are represented by x₆YxIx, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5, 6, 7 or 8 of the variable (i.e., x) positions. Incertain embodiments, the amino acid sequence x₆YxIx is not identical tothe amino acid sequence corresponding to amino acids 24-35 of SEQ IDNO: 1. Exemplary ¹⁰Fn3 designs having amino acids 24-35 represented byx₆YxIx include, e.g., NP4W, which is shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 52-55 of SEQ ID NO: 1or 6 are represented by x₄, wherein x is either any amino acid or x isany amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3 or 4 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₄ is not identical to the aminoacid sequence corresponding to amino acids 52-55 of SEQ ID NO: 1.Exemplary ¹⁰Fn3 designs having amino acids 52-55 represented by x₄include, e.g., Li-5 and LI-52, which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 47-55 of SEQ ID NO: 1or 6 are represented by xFxVx₄, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5 or 6 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence xFxVx₄ is not identical to theamino acid sequence corresponding to amino acids 47-55 of SEQ ID NO: 1.Exemplary ¹⁰Fn3 designs having amino acids 47-55 represented by xFxVx₄include, e.g., NP4W, which is shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 51-60 of SEQ ID NO: 1or 6 are represented by x₆AxIx, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5, 6, 7 or 8 of the variable (i.e., x) positions. Incertain embodiments, the amino acid sequence x₆AxIx is not identical tothe amino acid sequence corresponding to amino acids 51-60 of SEQ IDNO: 1. Exemplary ¹⁰Fn3 designs having amino acids 51-60 represented byx₆AxIx include, e.g., NP4E1, NP4E2 and NP4E3, which are shown in FIG.13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 77-83 of SEQ ID NO: 1or 6 are represented by x₃₋₁₂, such as x₇, wherein x is either any aminoacid or x is any amino acid except the amino acid that occurs at thecorresponding position in SEQ ID NO: 1 or 6. In certain embodiments, xis not the amino acid that occurs at the corresponding position in SEQID NO: 1 or 6 in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the variable (i.e.,x) positions. In certain embodiments, the amino acid sequence x₃₋₁₂ isnot identical to the amino acid sequence corresponding to amino acids77-83 of SEQ ID NO: 1 or 6. Exemplary ¹⁰Fn3 designs having amino acids77-83 represented by x₃₋₁₂ include, e.g., LI-3(a), LI-3(b), LI-5,LI-31(a), (c), LI-39F(b), LI-39F(f), LI-51, LI-52, LI-59(a)-(f), whichare shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 77-86 of SEQ ID NO: 1or 6 are represented by x₆₋₁₅, such as x₁₀, wherein x is either anyamino acid or x is any amino acid except the amino acid that occurs atthe corresponding position in SEQ ID NO: 1 or 6. In certain embodiments,x is not the amino acid that occurs at the corresponding position in SEQID NO: 1 or 6 in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the variable (i.e.,x) positions. In certain embodiments, the amino acid sequence x₆₋₁₅ isnot identical to the amino acid sequence corresponding to amino acids77-86 of SEQ ID NO: 1 or 6. Exemplary ¹⁰Fn3 designs having amino acids77-86 represented by x₆₋₁₅ include, e.g., LI-32(a)-(c), LI-39F(a),LI-39F(e), LI-53 and LI-54, which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 75-86 of SEQ ID NO: 1or 6 are represented by x₄₋₈Sx, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5 or 6 of the variable (i.e., x) positions. In certainembodiments, the amino acid sequence x₄₋₈Sx is not identical to theamino acid sequence corresponding to amino acids 75-86 of SEQ ID NO: 1or 6. Exemplary ¹⁰Fn3 designs having amino acids 75-86 represented byx₄₋₈Sx include, e.g., LI-38(a)-(c), LI-39(a)-(c), LI-39F(c) andLI-39F(g) which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 75-83 of SEQ ID NO: 1or 6 are represented by x₈₋₁₄, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5, 6 or 7 of the variable (i.e., x) positions. Incertain embodiments, the amino acid sequence x₈₋₁₄ is not identical tothe amino acid sequence corresponding to amino acids 75-83 of SEQ ID NO:1 or 6. Exemplary ¹⁰Fn3 designs having amino acids 75-83 represented byx₈₋₁₄ include, e.g., LI-38(d)-(f), LI-39(d)-(f), LI-39(j)-(l), LI-39F(d)and LI-39F(h), which are shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 75-89 of SEQ ID NO: 1or 6 are represented by xxx₃₋₁₂xIx, wherein x is either any amino acidor x is any amino acid except the amino acid that occurs at thecorresponding position in SEQ ID NO: 1 or 6. In certain embodiments, xis not the amino acid that occurs at the corresponding position in SEQID NO: 1 or 6 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of thevariable (i.e., x) positions. In certain embodiments, the amino acidsequence xxx₃₋₁₂xIx is not identical to the amino acid sequencecorresponding to amino acids 75-89 of SEQ ID NO: 1 or 6. Exemplary ¹⁰Fn3designs having amino acids 75-89 represented by xxx₃₋₁₂xIx include,e.g., NP4W, which is shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 76-86 of SEQ ID NO: 1or 6 are represented by xx₃₋₁₂, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the variable (i.e., x)positions. In certain embodiments, the amino acid sequence xx₃₋₁₂ is notidentical to the amino acid sequence corresponding to amino acids 76-86of SEQ ID NO: 1 or 6. Exemplary ¹⁰Fn3 designs having amino acids 76-86represented by xx₃₁₂ include, e.g., NP4E1, which is shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 76-86 of SEQ ID NO: 1or 6 are represented by xx₄₋₈Sx, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5, 6 or 7 of the variable (i.e., x) positions. Incertain embodiments, the amino acid sequence xx₄₋₈Sx is not identical tothe amino acid sequence corresponding to amino acids 76-86 of SEQ ID NO:1 or 6. Exemplary ¹⁰Fn3 designs having amino acids 76-86 represented byxx₄₋₈Sx include, e.g., NP4E2, which is shown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequencewherein the amino acids corresponding to residues 76-83 of SEQ ID NO: 1or 6 are represented by xx₈₋₁₄, wherein x is either any amino acid or xis any amino acid except the amino acid that occurs at the correspondingposition in SEQ ID NO: 1 or 6. In certain embodiments, x is not theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6 in 1, 2, 3, 4, 5, 6, 7 or 8 of the variable (i.e., x) positions. Incertain embodiments, the amino acid sequence xx₈₋₁₄ is not identical tothe amino acid sequence corresponding to amino acids 76-83 of SEQ ID NO:1 or 6. Exemplary ¹⁰Fn3 designs having amino acids 76-83 represented byxx₈₋₁₄ include, e.g., NP4E3, which is shown in FIG. 13.

¹⁰Fn3 domains may also comprise a combination of 2, 3 or 4 of the abovedescribed variable regions. Exemplary combinations of variable regionsinclude (i) an N-terminal variable region and a BC variable region; (ii)an N-terminal variable region and a DE variable region; (iii) anN-terminal variable region and an FG variable region; (iv) a BC variableregion and a DE variable region; (v) a BC variable region and an FGvariable region; and (vi) a DE variable region and an FG variableregion.

An N-terminal variable region may comprise, consist of, or consistessentially of:

(1) a variation of the amino acids corresponding to residues 1-9 of SEQID NO: 1 or 6, wherein the variation is represented by x₁₋₉Lx; or

(2) a variation of the amino acids corresponding to residues 1-9 of SEQID NO: 1 or 6, wherein the variation consists of the amino acid sequenceGVSDVPGGSG, or a portion thereof, linked to an amino acid sequencerepresented by x₁₋₉Lx,

wherein x is either any amino acid or x is any amino acid except theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6.

A BC variable region may comprise, consist of, or consist essentiallyof:

(1) a variation of the amino acids corresponding to residues 23-29 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₇; or

(2) a variation of the amino acids corresponding to residues 25-29 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₅; or

(3) a variation of the amino acids corresponding to residues 26-29 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₄; or

(4) a variation of the amino acids corresponding to residues 27-29 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₃; or

(5) a variation of the amino acids corresponding to residues 24-28 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₅; or

(6) a variation of the amino acids corresponding to residues 25-28 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₄; or

(7) a variation of the amino acids corresponding to residues 26-28 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₃; or

(8) a variation of the amino acids corresponding to residues 17-26 ofSEQ ID NO: 1 or 6, wherein the variation is represented by xLxIxWx₄; or

(9) a variation of the amino acids corresponding to residues 24-35 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₆YxIx,

wherein x is either any amino acid or x is any amino acid except theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6.

A DE variable region may comprise, consist of, or consist essentiallyof:

(1) a variation of the amino acids corresponding to residues 52-55 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₄; or

(2) a variation of the amino acids corresponding to residues 47-55 ofSEQ ID NO: 1 or 6, wherein the variation is represented by xFxVx₄; or

(3) a variation of the amino acids corresponding to residues 51-60 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₆AxIx,

wherein x is either any amino acid or x is any amino acid except theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6.

An FG variable region may comprise, consist of, or consist essentiallyof:

(1) a variation of the amino acids corresponding to residues 77-83 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₃₋₁₂; or

(2) a variation of the amino acids corresponding to residues 77-86 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₆₋₁₅; or

(3) a variation of the amino acids corresponding to residues 75-86 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₄₋₈Sx; or

(4) a variation of the amino acids corresponding to residues 75-83 ofSEQ ID NO: 1 or 6, wherein the variation is represented by x₈₋₁₄; or

(5) a variation of the amino acids corresponding to residues 75-89 ofSEQ ID NO: 1 or 6, wherein the variation is represented by xxx₃₋₁₂xIx;or

(6) a variation of the amino acids corresponding to residues 76-86 ofSEQ ID NO: 1 or 6, wherein the variation is represented by xx₃₋₁₂; or

(7) a variation of the amino acids corresponding to residues 76-86 ofSEQ ID NO: 1 or 6, wherein the variation is represented by xx₄₋₈Sx; or

(8) a variation of the amino acids corresponding to residues 76-83 ofSEQ ID NO: 1 or 6, wherein the variation is represented by xx₈₋₁₄,

wherein x is either any amino acid or x is any amino acid except theamino acid that occurs at the corresponding position in SEQ ID NO: 1 or6.

Exemplary combinations of N-terminal, BC, DE and FG variable regions areshown in FIG. 13.

In certain embodiments, a ¹⁰Fn3 domain comprises an amino acid sequenceset forth in FIG. 13 and does not comprise any other amino acidvariation, such an amino acid substitution, addition or deletion,outside of the variable regions (i.e., outside of the N-terminal, BC, DEand FG variable regions). In certain embodiments, a ¹⁰Fn3 domaincomprises an amino acid sequence set forth in FIG. 13 and at least, atmost or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30,35 or 40 amino acid variations, such as amino acid substitutions,additions or deletions, outside of the variable regions. In certainembodiments, a ¹⁰Fn3 domain comprises an amino acid sequence set forthin FIG. 13 and at least, at most or exactly 1-25, 1-20, 1-15, 1-10, 1-5,1-3, 1-2, 3-25, 3-20, 3-15, 3-10, 3-5, 5-25, 5-20, 5-15 or 5-10 aminoacid variations, such as amino acid substitutions, additions ordeletions, outside of the variable regions. A ¹⁰Fn3 domain may comprisean amino acid sequence that is at least 40%, 50%, 60%, 70%, 75%, 80%,85% or 90% identical to the amino acid sequence having SEQ ID NO: 1 or6, and comprising the amino acid variations of any one of the sequences(SEQ ID NOs: 73-131 or SEQ ID NOs: 76-131) set forth in FIG. 13. A ¹⁰Fn3domain may comprise the amino acid variations of an amino acid sequenceof any one of the sequences set forth in FIG. 13, wherein the regionsindicated as constant in the amino acid sequence comprise (consist of,or consist essentially of) an amino acid sequence that is at least 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence at the corresponding location in SEQ ID NO: 1or 6. The percentage identity may be calculated as an average numberbetween the percentage identity of several regions of a ¹⁰Fn3 sequence.For example, the percentage identity of the regions outside the variableregions of a ¹⁰Fn3 molecule may be calculated by obtaining the averagevalue of the percent identity of each individual region outside orbetween the variable regions. A ¹⁰Fn3 domain may comprise a variableregion, e.g., an N-terminal, BC, DE or FG variable region, and at most50, 40, 35, 30, 25, 20, 15, 10, 7, 5, 3, 2 or 1 additional amino acidchanges, such as amino acid substitutions, deletions or additionsrelative to SEQ ID NO: 1 or 6. A ¹⁰Fn3 domain may comprise a variableregion, e.g., an N-terminal, BC, DE or FG variable region, wherein theamino acid sequence of the ¹⁰Fn3 domain outside of the variable regionis at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identicalto the corresponding sequence in SEQ ID NO: 1 or 6. A ¹⁰Fn3 domain maycomprise a variable region, e.g., an N-terminal, BC, DE or FG variableregion, wherein the ¹⁰Fn3 domain comprises at least one amino acidvariation in the amino acid sequence corresponding to another variableregion.

A ¹⁰Fn3 domain may bind to a desired target with a K_(d) of less than500 nM, 100 nM, 50 nM, 1 nM, 500 pM, 100 pM or less. In someembodiments, a ¹⁰Fn3 domain binds to a desired target with a K_(d)between 1 pM and 1 μM, between 100 pM and 500 nM, between 1 nM and 500nM, or between 1 nM and 100 nM. In exemplary embodiments, a ¹Fn3 domainbinds specifically to a target that is not bound by a wild-type Fn3domain, particularly, the wild-type human ¹⁰Fn3 domain.

G. Multivalent Proteins

In certain embodiments, the fibronectin based scaffold protein is amultivalent protein that comprises two or more ¹⁰Fn3 domains. Forexample, a multivalent fibronectin based scaffold protein may comprise2, 3 or more ¹Fn3 domains that are covalently associated. In exemplaryembodiments, the fibronectin based scaffold protein is a bispecific ordimeric protein comprising two ¹⁰Fn3 domains. In certain embodiments, amultivalent fibronectin based protein scaffold comprises a first ¹⁰Fn3domain that binds to a first target molecule and a second ¹⁰Fn3 domainthat binds to a second target molecule. The first and second targetmolecules may be the same or different target molecules. When the firstand second target molecules are the same, the first and second ¹⁰Fn3domains may bind to the same target but at different epitopes.Additionally, when the first and second target molecules are the same,the regions of modification in the ¹⁰Fn3 domain that are associated withtarget binding may be the same or different. Furthermore, the first andsecond ¹⁰Fn3 domains may be based on the same or different scaffolddesigns. For example, a multivalent fibronectin based protein scaffoldmay comprise two ¹⁰Fn3 domains, wherein both ¹⁰Fn3 are based on the samenon-traditional scaffold design described herein, wherein one of the¹⁰Fn3 domains is based on a first type of non-traditional scaffolddesign and the second ¹Fn3 domain is based on a second type ofnon-traditional scaffold design, or one of the ¹Fn3 domains is based ona non-traditional scaffold design and the second is based on atraditional scaffold design (i.e., the BC, DE and FG loops aremodified).

In exemplary embodiments, each ¹⁰Fn3 domain of a multivalent fibronectinbased protein scaffold binds to a desired target with a K_(d) of lessthan 500 nM, 100 nM, 50 nM, 1 nM, 500 pM, 100 pM or less. In someembodiments, each ¹⁰Fn3 domain of a multivalent fibronectin basedprotein scaffold binds to a desired target with a K_(d) between 1 pM and1 μM, between 100 pM and 500 nM, between 1 nM and 500 nM, or between 1nM and 100 nM. In exemplary embodiments, each ¹Fn3 domain of amultivalent fibronectin based protein scaffold binds specifically to atarget that is not bound by a wild-type ¹⁰Fn3 domain, particularly thewild-type human ¹⁰Fn3 domain.

The ¹⁰Fn3 domains in a multivalent fibronectin based scaffold proteinmay be connected by a polypeptide linker. Exemplary polypeptide linkersinclude polypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3,or 1-2 amino acids. Suitable linkers for joining the ¹⁰Fn3 domains arethose which allow the separate domains to fold independently of eachother forming a three dimensional structure that permits high affinitybinding to a target molecule. Specific examples of suitable linkersinclude glycine-serine based linkers, glycine-proline based linkers,proline-alanine based linkers as well as linkers having the amino acidsequence of SEQ ID NO: 32. In some embodiments, the linker is aglycine-serine based linker. These linkers comprise glycine and serineresidues and may be between 8 and 50, 10 and 30, and 10 and 20 aminoacids in length. Examples of such linkers include SEQ ID NOs: 39-43. Insome embodiments, the linker is a glycine-proline based linker. Theselinkers comprise glycine and proline residues and may be between 3 and30, 10 and 30, and 3 and 20 amino acids in length. Examples of suchlinkers include SEQ ID NOs: 33-35. In some embodiments, the linker is aproline-alanine based linker. These linkers comprise proline and alanineresidues and may be between 3 and 30, 10 and 30, 3 and 20 and 6 and 18amino acids in length. Examples of such linkers include SEQ ID NOs:36-38. In exemplary embodiments, the linker does not contain any Asp-Lys(DK) pairs.

Pharmacokinetic Moieties

In one aspect, the application provides for fibronectin based scaffoldproteins further comprising a pharmacokinetic (PK) moiety.Pharmokinetics encompasses properties of a compound including, by way ofexample, absorption, distribution, metabolism, and elimination by asubject. Improved pharmacokinetics may be assessed according to theperceived therapeutic need. Often it is desirable to increasebioavailability and/or increase the time between doses, possibly byincreasing the time that a protein remains available in the serum afterdosing. In some instances, it is desirable to improve the continuity ofthe serum concentration of the protein over time (e.g., decrease thedifference in serum concentration of the protein shortly afteradministration and shortly before the next administration). Thefibronectin based scaffold proteins may be attached to a moiety thatreduces the clearance rate of the polypeptide in a mammal (e.g., mouse,rat, or human) by greater than three-fold relative to the unmodifiedpolypeptide. Other measures of improved pharmacokinetics may includeserum half-life, which is often divided into an alpha phase and a betaphase. Either or both phases may be improved significantly by additionof an appropriate moiety. A PK moiety refers to any protein, peptide, ormoiety that affects the pharmacokinetic properties of a biologicallyactive molecule when fused to the biologically active molecule.

PK moieties that tend to slow clearance of a protein from the bloodinclude polyoxyalkylene moieties, e.g., polyethylene glycol, sugars(e.g., sialic acid), and well-tolerated protein moieties (e.g., Fc, Fcfragments, transferrin, or serum albumin). The fibronectin basedscaffold proteins may be fused to albumin or a fragment (portion) orvariant of albumin as described in U.S. Publication No. 20070048282. Insome embodiments, the PK moiety is a serum albumin binding protein suchas those described in U.S. Publication Nos. 2007/0178082 and2007/0269422. In some embodiments, the PK moiety is a serumimmunoglobulin binding protein such as those described in U.S.Publication No. 2007/0178082.

In some embodiments, the fibronectin based scaffold proteins may beattached to a PK moiety comprising a nonproteinaceous polymer. In someembodiments, the polymer is polyethylene glycol (“PEG”), polypropyleneglycol, or polyoxyalkylenes, as described in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In exemplaryembodiments, the polymer is a PEG moiety.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula: X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH(1), where n is 20 to 2300 and X is H or a terminal modification, e.g.,a C₁₋₄ alkyl. In one embodiment, the PEG of the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). APEG can contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462. One form of PEGsincludes two PEG side-chains (PEG2) linked via the primary amino groupsof a lysine (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol.Chem., 252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a fibronectin based scaffold proteincontaining a PEG molecule is also known as a conjugated protein, whereasthe protein lacking an attached PEG molecule can be referred to asunconjugated.

The size of PEG utilized will depend on several factors including theintended use of the fibronectin based scaffold protein. Larger PEGs arepreferred to increase half life in the body, blood, non-bloodextracellular fluids or tissues. For in vivo cellular activity, PEGs ofthe range of about 10 to 60 kDa are preferred, as well as PEGs less thanabout 100 kDa and more preferably less than about 60 kDa, though sizesgreater than about 100 kDa can be used as well. For in vivo imagingapplications, smaller PEGs, generally less than about 20 kDa, may beused that do not increase half life as much as larger PEGs so as topermit quicker distribution and less half life. A variety of molecularmass forms of PEG can be selected, e.g., from about 1,000 Daltons (Da)to 100,000 Da (n is 20 to 2300), for conjugating to fibronectin basedscaffold proteins. The number of repeating units “n” in the PEG isapproximated for the molecular mass described in Daltons. It ispreferred that the combined molecular mass of PEG on an activated linkeris suitable for pharmaceutical use. Thus, in one embodiment, themolecular mass of the PEG molecules does not exceed 100,000 Da. Forexample, if three PEG molecules are attached to a linker, where each PEGmolecule has the same molecular mass of 12,000 Da (each n is about 270),then the total molecular mass of PEG on the linker is about 36,000 Da(total n is about 820). The molecular masses of the PEG attached to thelinker can also be different, e.g., of three molecules on a linker twoPEG molecules can be 5,000 Da each (each n is about 110) and one PEGmolecule can be 12,000 Da (n is about 270). In some embodiments, one PEGmoiety is conjugated to the fibronectin based scaffold protein. In someembodiments, the PEG moiety is about 20, 30, 40, 50, 60, 70, 80, or 90KDa. In some embodiments, the PEG moiety is about 40 KDa.

In some embodiments, PEGylated fibronectin based scaffold proteinscontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin a pegylated fibronectin based scaffold protein is from about 3,000 Dato 60,000 Da, or from about 10,000 Da to 36,000 Da. In a one embodiment,the PEG in a pegylated fibronectin based scaffold protein is asubstantially linear, straight-chain PEG.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated fibronectin based scaffold protein willbe used therapeutically, the desired dosage, circulation time,resistance to proteolysis, immunogenicity, and other considerations. Fora discussion of PEG and its use to enhance the properties of proteins,see N. V. Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993).

In some embodiments, a fibronectin based scaffold protein is covalentlylinked to one poly(ethylene glycol) group of the formula: —CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of the poly(ethyleneglycol) group forming an amide bond with one of the amino groups of thebinding polypeptide; R being lower alkyl; x being 2 or 3; m being fromabout 450 to about 950; and n and m being chosen so that the molecularweight of the conjugate minus the binding polypeptide is from about 10to 40 kDa. In one embodiment, a fibronectin based scaffold protein'sε-amino group of a lysine is the available (free) amino group.

In one specific embodiment, carbonate esters of PEG are used to form thePEG-fibronectin based scaffold protein conjugates.N,N′-disuccinimidylcarbonate (DSC) may be used in the reaction with PEGto form active mixed PEG-succinimidyl carbonate that may be subsequentlyreacted with a nucleophilic group of a linker or an amino group of afibronectin based scaffold protein (see U.S. Pat. Nos. 5,281,698 and5,932,462). In a similar type of reaction,1,1′-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may bereacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixedcarbonate (U.S. Pat. No. 5,382,657), respectively.

Pegylation of a fibronectin based scaffold protein can be performedaccording to the methods of the state of the art, for example byreaction of the fibronectin based scaffold protein withelectrophilically active PEGs (supplier: Shearwater Corp., USA, worldwide web at shearwatercorp.com). Preferred PEG reagents of the presentinvention are, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA),butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., et al.,Bioconjugate Chem. 6 (1995) 62-69). Such methods may used to pegylate atan ε-amino group of a lysine of a fibronectin based scaffold protein orat the N-terminal amino group of the fibronectin based scaffold protein.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a fibronectin based scaffold protein (Sartore, L., et al., Appl.Biochem. Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7,363-368 (1996); Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat.No. 5,766,897). U.S. Pat. Nos. 6,610,281 and 5,766,897 describesexemplary reactive PEG species that may be coupled to sulfhydryl groups.

In some embodiments, the pegylated fibronectin based scaffold protein isproduced by site-directed pegylation, particularly by conjugation of PEGto a cysteine moiety. In certain embodiments, the Cys residue may bepositioned at the N-terminus, between the N-terminus and the mostN-terminal beta or beta-like strand, at the C-terminus, or between theC-terminus and the most C-terminal beta or beta-like strand of thefibronectin based scaffold protein. A Cys residue may be situated atother positions as well, particularly any of the loops that do notparticipate in target binding or between two binding domains of amultivalent fibronectin based scaffold protein. A PEG moiety may also beattached by other chemistry, including by conjugation to amines.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a fibronectin based scaffold protein, the cysteine residuesare native to the fibronectin based scaffold protein, whereas in otherembodiments, one or more cysteine residues are engineered into thefibronectin based scaffold protein. Mutations may be introduced into afibronectin based scaffold protein coding sequence to generate cysteineresidues. This might be achieved, for example, by mutating one or moreamino acid residues to cysteine. Preferred amino acids for mutating to acysteine residue include serine, threonine, alanine and otherhydrophilic residues. Preferably, the residue to be mutated to cysteineis a surface-exposed residue. Algorithms are well-known in the art forpredicting surface accessibility of residues based on primary sequenceor a protein. Alternatively, surface residues may be predicted bycomparing the amino acid sequences of fibronectin based scaffoldproteins, given that the crystal structure of the tenth fn3 domainframework based on which fibronectin based scaffold proteins aredesigned has been solved (see Dickinson, et al., J. Mol. Biol. 236(4):1079-92 (1994)) and thus the surface-exposed residues identified. In oneembodiment, cysteine residues are introduced into fibronectin basedscaffold protein at or near the N- and/or C-terminus, or within loopregions. Pegylation of cysteine residues may be carried out using, forexample, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, orPEG-orthopyridyl disulfide.

In some embodiments, the pegylated fibronectin based scaffold proteincomprises a PEG molecule covalently attached to the alpha amino group ofthe N-terminal amino acid. Site specific N-terminal reductive aminationis described in Pepinsky et al., (2001) JPET, 297,1059, and U.S. Pat.No. 5,824,784. The use of a PEG-aldehyde for the reductive amination ofa protein utilizing other available nucleophilic amino groups isdescribed in U.S. Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol.Chem. 254,12579, and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated fibronectin based scaffold proteinscomprise one or more PEG molecules covalently attached to a linker,which in turn is attached to the alpha amino group of the amino acidresidue at the N-terminus of the fibronectin based scaffold protein.Such an approach is disclosed in U.S. Publication No. 2002/0044921 andPCT Publication No. WO94/01451.

In one embodiment, a fibronectin based scaffold protein is pegylated atthe C-terminus. In a specific embodiment, a protein is pegylated at theC-terminus by the introduction of C-terminal azido-methionine and thesubsequent conjugation of a methyl-PEG-triarylphosphine compound via theStaudinger reaction. This C-terminal conjugation method is described inCazalis et al., C-Terminal Site-Specific PEGylation of a TruncatedThrombomodulin Mutant with Retention of Full Bioactivity, BioconjugChem. 2004; 15(5):1005-1009.

In exemplary embodiments, a fibronectin based scaffold protein ispegylated in a C-terminal tail region as described further herein. Inexemplary embodiments, the C-terminal contains a Cys residue, which isused as the site of attachment for the PEG moiety. Exemplary C-terminaltails include, for example, a polypeptide having any one of SEQ ID NOs:23, 24 or 31.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated fibronectin based scaffold proteins, such assize exclusion (e.g., gel filtration) and ion exchange chromatography.Products may also be separated using SDS-PAGE. Products that may beseparated include mono-, di-, tri- poly- and un-pegylated fibronectinbased scaffold proteins, as well as free PEG. The percentage of mono-PEGconjugates can be controlled by pooling broader fractions around theelution peak to increase the percentage of mono-PEG in the composition.About ninety percent mono-PEG conjugates represents a good balance ofyield and activity. Compositions in which, for example, at leastninety-two percent or at least ninety-six percent of the conjugates aremono-PEG species may be desired. In an embodiment of this invention thepercentage of mono-PEG conjugates is from ninety percent to ninety-sixpercent.

In one embodiment of the invention, the PEG in a pegylated fibronectinbased scaffold protein is not hydrolyzed from the pegylated amino acidresidue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5)over 8 to 16 hours at room temperature, and is thus stable. In oneembodiment, greater than 80% of the composition is stablemono-PEG-fibronectin based scaffold protein, more preferably at least90%, and most preferably at least 95%.

In another embodiment, the pegylated fibronectin based scaffold proteinswill preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%,95% or 100% of the biological activity associated with the unmodifiedprotein. In one embodiment, biological activity refers to its ability tobind to one or more target molecules, as assessed by K_(d), k_(on) ork_(off). In one specific embodiment, the pegylated fibronectin basedscaffold protein shows an increase in binding to one or more targetmolecules relative to unpegylated fibronectin based scaffold protein.

The serum clearance rate of PEG-modified fibronectin based scaffoldproteins may be decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or even 90%, relative to the clearance rate of the unmodifiedfibronectin based scaffold protein. The PEG-modified fibronectin basedscaffold protein may have a half-life (t_(1/2)) which is enhancedrelative to the half-life of the unmodified fibronectin based scaffoldprotein. The half-life of PEG-modified fibronectin based scaffoldprotein may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, oreven by 1000% relative to the half-life of the unmodified fibronectinbased scaffold protein. In some embodiments, the protein half-life isdetermined in vitro, such as in a buffered saline solution or in serum.In other embodiments, the protein half-life is an in vivo half life,such as the half-life of the fibronectin based scaffold protein in theserum or other bodily fluid of an animal.

Nucleic Acid-Protein Fusion Technology

In one aspect, the application provides fibronectin based scaffoldproteins comprising a fibronectin type III domain that bind a humantarget, such as, for example, TNF-alpha, DLL4, IL-17, PXR or otherproteins. One way to rapidly make and test Fn3 domains with specificbinding properties is the nucleic acid-protein fusion technology ofAdnexus, a Bristol-Myers Squibb Company. Such in vitro expression andtagging technology, termed PROfusion™, that exploits nucleicacid-protein fusions (RNA- and DNA-protein fusions) may be used toidentify novel polypeptides and amino acid motifs that are important forbinding to proteins. Nucleic acid-protein fusion technology is atechnology that covalently couples a protein to its encoding geneticinformation. For a detailed description of the RNA-protein fusiontechnology and fibronectin-based scaffold protein library screeningmethods see Szostak et al., U.S. Pat. Nos. 6,258,558; 6,261,804;6,214,553; 6,281,344; 6,207,446; 6,518,018; PCT Publication Nos.WO00/34784; WO01/64942; WO02/032925; and Roberts and Szostak, Proc Natl.Acad. Sci. 94:12297-12302, 1997, herein incorporated by reference.

Vectors & Polynucleotides Embodiments

Nucleic acids encoding any of the various fibronectin based scaffoldproteins disclosed herein may be synthesized chemically, enzymaticallyor recombinantly. Codon usage may be selected so as to improveexpression in a cell. Such codon usage will depend on the cell typeselected. Specialized codon usage patterns have been developed for E.coli and other bacteria, as well as mammalian cells, plant cells, yeastcells and insect cells. See for example: Mayfield et al., Proc Natl AcadSci USA. 2003 January 21; 100(2):438-42; Sinclair et al. Protein ExprPurif. 2002 October; 26(1):96-105; Connell N D. Curr Opin Biotechnol.2001 October; 12(5):446-9; Makrides et al. Microbiol Rev. 1996September; 60(3):512-38; and Sharp et al. Yeast. 1991 October;7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The fibronectin based scaffold proteins described herein may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process a native signal sequence, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, 1 pp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces alpha-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in PCT Publication No. WO90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor regions may be ligated in readingframe to DNA encoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the fibronectin-based scaffold protein. Promoters suitablefor use with prokaryotic hosts include the phoA promoter, beta-lactamaseand lactose promoter systems, alkaline phosphatase, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the fibronectin based scaffoldprotein.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP Patent Publication No. 73,657. Yeast enhancers also areadvantageously used with yeast promoters.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human .beta.-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus.Alternatively, the rous sarcoma virus long terminal repeat can be usedas the promoter.

Transcription of a DNA encoding fibronectin based scaffold proteins byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, .alpha.-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thepolypeptide-encoding sequence, but is preferably located at a site 5′from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the polypeptide. Oneuseful transcription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the fibronectin based scaffold protein.Examples of protein tags include but are not limited to a histidine tag,a FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts can be found in Cloning Vectors: A Laboratory Manual,(Elsevier, New York, 1985), the relevant disclosure of which is herebyincorporated by reference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47,1988). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified fibronectin based scaffold proteinsare prepared by culturing suitable host/vector systems to express therecombinant proteins. For many applications, the small size of thefibronectin based scaffold proteins would make expression in E. coli thepreferred method for expression. The fibronectin based scaffold proteinis then purified from culture media or cell extracts.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the fibronectin based scaffold proteinsmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),(Sigma)) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO87/00195; or U.S. Pat.No. Re. 30,985 may be used as culture media for the host cells. Any ofthese media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Fibronectin based scaffold proteins disclosed herein can also beproduced using cell-free translation systems. For such purposes thenucleic acids encoding the fibronectin based scaffold protein must bemodified to allow in vitro transcription to produce mRNA and to allowcell-free translation of the mRNA in the particular cell-free systembeing utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system).

Fibronectin based scaffold proteins can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).Modifications to the fibronectin based scaffold protein can also beproduced by chemical synthesis.

The fibronectin based scaffold proteins disclosed herein can be purifiedby isolation/purification methods for proteins generally known in thefield of protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, fibronectin based scaffold proteins may beexchanged into different buffers and/or concentrated by any of a varietyof methods known to the art, including, but not limited to, filtrationand dialysis.

The purified fibronectin based scaffold protein is preferably at least85% pure, more preferably at least 95% pure, and most preferably atleast 98% pure. Regardless of the exact numerical value of the purity,the fibronectin based scaffold protein is sufficiently pure for use as apharmaceutical product.

Exemplary Uses

In one aspect, the application provides fibronectin based scaffoldproteins labeled with a detectable moiety. The fibronectin basedscaffold proteins may be used for a variety of diagnostic applications.The detectable moiety can be any one which is capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as H3, C14, C13, P32, S35,or I131; a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for conjugating a protein to the detectablemoiety may be employed, including those methods described by Hunter, etal., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974);Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982). In vitro methods, include conjugationchemistry well know in the art including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a detectable moiety to a fibronectin basedscaffold protein, a linking group or reactive group is used. Suitablelinking groups are well known in the art and include disulfide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups and esterase labile groups. Preferred linking groups aredisulfide groups and thioether groups depending on the application. Forpolypeptides without a Cys amino acid, a Cys can be engineered in alocation to allow for activity of the protein to exist while creating alocation for conjugation.

Fibronectin based scaffold proteins linked with a detectable moiety areuseful for in vitro or in vivo imaging. The polypeptide may be linked toa radio-opaque agent or radioisotope, administered to a subject,preferably into the bloodstream, and the presence and location of thelabeled protein in the subject may be assayed. This imaging technique isuseful, for example, in the staging and treatment of malignancies whenthe fibronectin based scaffold protein binds to a target associated withcancer. The fibronectin based scaffold protein may be labeled with anymoiety that is detectable in a subject, whether by nuclear magneticresonance, radiology, or other detection means known in the art.

Fibronectin based scaffold proteins also are useful as affinitypurification agents. In this process, the fibronectin based scaffoldproteins are immobilized on a suitable support, such as Sephadex resinor filter paper, using methods well known in the art.

Fibronectin based scaffold proteins can be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)).

In certain aspects, the disclosure provides methods for detecting atarget molecule in a sample. A method may comprise contacting the samplewith a fibronectin based scaffold protein described herein, wherein saidcontacting is carried out under conditions that allow fibronectin basedscaffold protein-target complex formation; and detecting said complex,thereby detecting said target in said sample. Detection may be carriedout using any technique known in the art, such as, for example,radiography, immunological assay, fluorescence detection, massspectroscopy, or surface plasmon resonance. The sample will often by abiological sample, such as a biopsy, and particularly a biopsy of atumor, or a suspected tumor, where the fibronectin based scaffoldprotein binds to a target associated with cancer. The sample may be froma human or other mammal. The fibronectin based scaffold protein may belabeled with a labeling moiety, such as a radioactive moiety, afluorescent moiety, a chromogenic moiety, a chemiluminescent moiety, ora hapten moiety. The fibronectin based scaffold protein may beimmobilized on a solid support.

In one aspect, the application provides fibronectin based scaffoldproteins useful in the treatment of disorders. The diseases or disordersthat may be treated will be dictated by the binding specificity of thefibronectin based scaffold protein. As described herein, fibronectinbased scaffold proteins may be designed to bind to any target ofinterest. Exemplary targets include, for example, TNF-alpha, DLL4, IL-17and PXR. Merely as an example, fibronectin based scaffold proteins thatbind to TNF-alpha may be used to treat autoimmune disorders such asrheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma;fibronectin based scaffold proteins that bind to IL-17 may be used totreat asthma; and fibronectin based scaffold proteins that bind to DLL4may be used to treat hyperproliferative disorders or diseases associatedwith unwanted angiogenesis, such as cancers or tumors.

The application also provides methods for administering fibronectinbased scaffold proteins to a subject. In some embodiments, the subjectis a human. In some embodiments, the fibronectin based scaffold proteinsare pharmaceutically acceptable to a mammal, in particular a human. A“pharmaceutically acceptable” composition refers to a composition thatis administered to an animal without significant adverse medicalconsequences. Examples of pharmaceutically acceptable compositionsinclude compositions comprising ¹⁰Fn3 domains that lack theintegrin-binding domain (RGD) and compositions that are essentiallyendotoxin or pyrogen free or have very low endotoxin or pyrogen levels.

Formulation and Administration

The application further provides pharmaceutically acceptablecompositions comprising the fibronectin based scaffold proteinsdescribed herein, wherein the composition is essentially endotoxinand/or pyrogen free.

Therapeutic formulations comprising fibronectin based scaffold proteinsare prepared for storage by mixing the described proteins having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyidimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The fibronectin based scaffold proteins may also be entrapped inmicrocapsule prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethyl cellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the fibronectin based scaffold proteinsdescribed herein, which matrices are in the form of shaped articles,e.g., films, or microcapsule. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated proteins remain inthe body for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

While the skilled artisan will understand that the dosage of eachfibronectin based scaffold protein will be dependent on the identity ofthe protein, the preferred dosages can range from about 10 mg/squaremeter to about 2000 mg/square meter, more preferably from about 50mg/square meter to about 1000 mg/square meter.

For therapeutic applications, the fibronectin based scaffold proteinsare administered to a subject, in a pharmaceutically acceptable dosageform. They can be administered intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. The protein may also be administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. Suitable pharmaceuticallyacceptable carriers, diluents, and excipients are well known and can bedetermined by those of skill in the art as the clinical situationwarrants. Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, pH about 7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The methods of thepresent invention can be practiced in vitro, in vivo, or ex vivo.

Administration of fibronectin based scaffold proteins, and one or moreadditional therapeutic agents, whether co-administered or administeredsequentially, may occur as described above for therapeutic applications.Suitable pharmaceutically acceptable carriers, diluents, and excipientsfor co-administration will be understood by the skilled artisan todepend on the identity of the particular therapeutic agent beingco-administered.

When present in an aqueous dosage form, rather than being lyophilized,the fibronectin based scaffold protein typically will be formulated at aconcentration of about 0.1 mg/ml to 100 mg/ml, although wide variationoutside of these ranges is permitted. For the treatment of disease, theappropriate dosage of fibronectin based scaffold proteins will depend onthe type of disease to be treated, the severity and course of thedisease, whether the fibronectin based scaffold proteins areadministered for preventive or therapeutic purposes, the course ofprevious therapy, the patient's clinical history and response to thefibronectin based scaffold protein, and the discretion of the attendingphysician. The fibronectin based scaffold protein is suitablyadministered to the patient at one time or over a series of treatments.

Fibronectin based scaffold proteins may also be used as crystallizationchaperones to generate structures of a protein with a compound ofinterest. For example, a fibronectin based scaffold protein thatspecifically binds to human pregnane X receptor (PXR) may be used as acrystallization chaperone to facilitate crystallization of a compoundwith PXR, e.g., the ligand binding domain (LBD) of PXR. Several ¹⁰Fn3molecules that bind to human PXR are described herein.

PXR activation upregulates cellular levels of several drug metabolizingenzymes such as cytochrome P450 enzymes and MDR1. The increasedexpression of CYP enzymes can alter the pharmacokinetics of drug andlead to dangerous drug-drug interactions including loss of therapeuticefficacy and increased toxicity. To avoid late stage clinical failuresand high costs associated with bringing a new drug to market, manypharmaceutical companies have adopted screening assays for earlydetection of compounds that activate PXR. Additionally in silicoscreening using known crystal structures of PXR are increasingly beingused to predict potential PXR activity. The large and flexible ligandbinding pocket of PXR and the potential of these compounds to bind todifferent locations and in multiple orientations within the ligandbinding cavity of PXR complicates reliable prediction of PXR activity.This is especially true for more advanced compounds/chemotypes withdesirable efficacy, selectivity and bioavailability towards the intendedtherapeutic target but with known PXR liability. Given theselimitations, a co-crystal structure is often required to define exactbinding interactions and to suggest specific modifications that candisturb crucial interactions related to PXR binding while maintainingactivity against the primary target. For example, in certainembodiments, a method for analyzing the interaction of a test agent withPXR comprises incubating together (i) PXR, or a ligand binding domainthereof; (ii) the test agent and (iii) a ¹⁰Fn3 protein specificallybinding to PXR, e.g., PXR LBD, under conditions suitable forcrystallization. Exemplary ¹⁰Fn3 proteins specifically binding to PXRLBD comprise an amino acid sequence that is at least 70%, 80%, 90%, 95%,97%, 98% or 99% identical to one of SEQ ID NOs: 48, 49 and 62-69 and 72.In certain embodiments, ¹⁰Fn3 proteins specifically binding to PXR LBDcomprise an amino acid sequence that differs from any one of SEQ ID NOs:48, 49 and 62-69 and 72 in at most 1, 2, 3, 5, 10, 15, 20 or 25 aminoacid changes, e.g., substitutions (such as conservative substitutions),additions or deletions. The method may further comprise inducingcrystallization and determining which portions (or atoms) of the testagent interacts with PXR (generally the ligand binding domain of PXR),and optionally modifying the test agent such that it no longer interactswith PXR or does so with lower affinity.

SEQUENCES Wild-Type ¹⁰Fn3 Sequences: WT ¹⁰Fn3 DomainVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSQ (SEQ ID NO: 1)WT ¹⁰Fn3 Domain Core Sequence version 1LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINY (SEQ ID NO: 2)WT ¹⁰Fn3 Domain with D80E SubstitutionVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPISINYRTEIDKPSQ (SEQ ID NO: 59)WT ¹⁰Fn3 Domain Core Sequence version 2EVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 60)WT ¹⁰Fn3 Domain Core Sequence version 3 VSDVPRD LEVVAA (X)_(n) LLISW(X)_(v) YRITY (X)_(w) FTV (X)_(x) ATISGL (X)_(y) YTITVYA (X)_(z) ISINYRT (SEQ ID NO: 22) WT ¹⁰Fn3 Domain Core Sequence version 4VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 6)DLL4 Binding WS-LI1 Binders:MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEQHSKYPHQEFTVPGSKSTATISGLKPGVDYTITVYAVTIQPQDPEQDYQYHYYETSSKPISINYRTEIDKPSQ (SEQ ID NO: 3)MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEHVADHFDHNQEFTVPGSKSTATISGLKPGVDYTITVYAVTYQFQDPEEHYYYHFYDSSSKPISINYRTEIDKPSQ (SEQ ID NO: 4)MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEYHEHYHSPGFSQKYHYEQEFTVPGSKSTATISGLKPGVDYTITVYAVTGHKHYHYYYYYHHESSKPISINYRTEIDKPSQ (SEQ ID NO: 5)Exemplary N-terminal Extension Sequences: MGVSDVPRDL (SEQ ID NO: 9)VSDVPRDL (SEQ ID NO: 10) GVSDVPRDL (SEQ ID NO: 11)X_(n)SDVPRDL, wherein n = 0, 1 or 2 amino acids,wherein when n = 1, X is Met or Gly, and whenn = 2, X is Met-Gly (SEQ ID NO: 16)X_(n)DVPRDL, wherein n = 0, 1 or 2 amino acids,wherein when n = 1, X is Met or Gly, and whenn = 2, X is Met-Gly (SEQ ID NO: 17)X_(n)VPRDL, wherein n = 0, 1 or 2 amino acids,wherein when n = 1, X is Met or Gly, and whenn = 2, X is Met-Gly (SEQ ID NO: 18)X_(n)PRDL, wherein n = 0, 1 or 2 amino acids,wherein when n = 1, X is Met or Gly, and whenn = 2, X is Met-Gly (SEQ ID NO: 19)X_(n)RDL, wherein n = 0, 1 or 2 amino acids,wherein when n = 1, X is Met or Gly, and whenn = 2, X is Met-Gly (SEQ ID NO: 20)X_(n)DL, wherein n = 0, 1 or 2 amino acids,wherein when n = 1, X is Met or Gly, and whenn = X is Met-Gly (SEQ ID NO: 21) MASTSG (SEQ ID NO: 50)Exemplary C-Terminal Tail Sequences: EIEK (SEQ ID NO: 7)EIEKPC (SEQ ID NO: 8) EGSGC (SEQ ID NO: 23) EIEKPCQ (SEQ ID NO: 24)EIEKPSQ (SEQ ID NO: 25) EIEKP (SEQ ID NO: 26) EIEKPS (SEQ ID NO: 27)EGSGS (SEQ ID NO: 28) EIDK (SEQ ID NO: 29) EIDKPSQ (SEQ ID NO: 30)EIDKPCQ (SEQ ID NO: 31) Exemplary Linker Sequences:PSTSTST (SEQ ID NO: 32) GPG (SEQ ID NO: 33) GPGPGPG (SEQ ID NO: 34)GPGPGPGPGPG (SEQ ID NO: 35) PAPAPA (SEQ ID NO: 36)PAPAPAPAPAPA (SEQ ID NO: 37) PAPAPAPAPAPAPAPAPA (SEQ ID NO: 38)GSGSGSGSGS (SEQ ID NO: 39) GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 40)GGGGSGGGGSGGGGS (SEQ ID NO: 41)GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42)GGGGSGGGGSGGGSG (SEQ ID NO: 43) 6XHis Tag: HEIHHHH (SEQ ID NO: 44)IL-17 Binding WS-LI1 Binders:MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEYHAFFASNGKYYFYIQEFTVPGSKSTATISGLKPGVDYTITVYAVTDDTVHHGDSNYHSSKPISINYRTEIDKPSQ (SEQ ID NO: 45)MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEYSSFFQHQGQYYHYIQEFTVPGSKSTATISGLKPGVDYTITVYAVTQHEHSQDSSKPISINYRTEIDKPSQ (SEQ ID NO: 46)MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEFSQFVHSDGEYYQEYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGQYDQDDEPSSKPISINYRTEIDKPSQ (SEQ ID NO: 47) PXR Binding WS1 Binders:MASTSGVSDVPRDLEVVAATPTSLLISWDAPAVPVSKYVIYYWPGALISSMQAFKVPGSKSTATISGLKPGVLYSIVVDALTGDGQGSYVWDPITITYRTEG SGS (SEQ ID NO: 48)MASTSGVSDVPRDLEVVAATPTSLLISWDAPAVTVHSYYITYQELQHHSVPQGFQVPGSKSTATISGLKPGVAYQIAVYAFTGPGLPPSDAPPIVIYYRTEG SGS (SEQ ID NO: 49)¹⁰Fn3 Loop and Scaffold Region Peptides from Figure 4:PTSLLISWDAPAVTVRYYRITYG (SEQ ID NO: 58)PVQEFTVPGSKSTATISGLK (SEQ ID NO: 51)TITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 52)MGEVVAATPTSLLIS(SEQ ID NO: 53) PHFPTRYYRITYGETGGNS (SEQ ID NO: 54)BC Loop Sequences from Example 2:PTSLLISWDAPAVTVRYYRITYG (SEQ ID NO: 55)PTSLLISWSARLKVARYYRITYG (SEQ ID NO: 56)PTSLLISWRHPHFPTRYYRITYG (SEQ ID NO: 57)IGF-1R Binding ¹⁰Fn3 Domain with Modified BC, DE and FG Loops:GVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQ (SEQ ID NO: 61)PXR Binding ¹⁰Fn3 molecules:The amino acid sequences of Adnectins 1-8 of FIG. 10 correspond to SEQID NOs: 70, 71, 62, 63, 72, 13, 14 and 15, respectively. Amino acidsequences of Adnectins 1-8 of FIG. 10 without the 6xHis tail (SEQ ID NO:44) correspond to SEQ ID NOs: 64, 65, 48, 49, 66, 67, 68 and 69,respectively.

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

Example 1. Expression and Purification of Fibronectin Based ScaffoldProteins

Selected binders were cloned into a PET9d vector and transformed into E.coli BL21 DE3 plysS cells. Transformed cells were inoculated in 5 ml LBmedium containing 50 μg/mL kanamycin and 34 μg/ml chloromphenicol in a24-well format and grown at 37° C. overnight (inoculums culture).Production cultures were established by aspirating 200 μl of theinoculum culture into 5 ml (in a 24-well format) of TB-OvernightExpression Media (auto induction) containing 50 μg/ml Kanamycin and 34μg/ml chloromphenicol. The cultures were grown at 37° C. for 4 hours atwhich time the temperature was lowered to 18° C. and grown for 20 hours.Cultures were harvested by centrifugation for 10 minutes at 2750 g at 4°C.

Cell pellets (in 24-well format) were lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1mg/ml lysozyme, 30 μg/ml DNAse, 2 μg/ml aprotonin, pH 8.0) and shaken atroom temperature for 1-3 hours. Lysates were clarified and re-rackedinto a 96-well format by transfer into a 96-well Whatman GF/D Unifilterfitted with a 96-well, 1.2 ml catch plate and filtered by positivepressure. The clarified lysates were transferred to a 96-well HisPurCobalt Plate that had been equilibrated with equilibration buffer (50 mMNaH₂PO₄, 0.5 M NaCl, 40 mM Imidazole, pH 8.0) and were incubated for 5min. Unbound material was removed by positive pressure. The resin waswashed 2×0.3 ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5mM CHAPS, 40 mM Imidazole, pH 8.0) with each wash removed by positivepressure. Prior to elution each well was washed with 50 μl Elutionbuffer (PBS+20 mM EDTA), incubated for 5 min and this wash was discardedby positive pressure. Protein was eluted by applying an additional 100μl of Elution buffer to each well. After a 30 minute incubation at roomtemperature the plate(s) were centrifuged for 5 minutes at 200 g andeluted protein was collected in 96-well catch plates containing 5 μl of0.5M MgCl₂ added to the bottom of elution catch plate prior to elution.Eluted protein was quantified using a total protein assay with SGE asthe protein standard. SGE is a wild-type ¹⁰Fn3 domain in which the RGDsequence in the FG loop is changed to SGE.

Example 2. Characterization of the Immunogenicity of 10Fn3 DomainPolypeptides

The adaptive immune response is initiated by the processing anddigestion of an internalized protein by an antigen-presenting cell(APC), such as a dendritic cell. The APC clips the internalized proteininto short peptides and then displays the peptides on its surface MHCClass II molecules. The peptide binding site of the MHC Class IImolecule is long and narrow, like a hot-dog bun, and holds its peptidein an extended format, with room for nine amino acids in the primarybinding site (and generally allows for short tails on either side of thepeptide). Certain pockets in the MHC binding site are dominant indetermining peptide binding. These pockets correspond to amino acidpositions 1, 4, 6, and 9 in the anchored portion of the 9-mer peptide. Apeptide that has favorable side chains at each of these four positionswill in general bind to HLA (an MHC Class II molecule) well.

Position 1 is thought to be the most important ‘anchor residue’ involvedin binding between the peptide and the HLA molecule. Position 1generally favors a hydrophobic side chain—thus, 9-mers that often bindHLA are initiated with V, I, L, M, F, Y, or W. The other positions aremuch more variable, with different HLA alleles favoring different setsof amino acids at each site. The immunogenicity of the polypeptidesdescribed herein were assessed using both in vitro and in silicomethods.

A—In Vitro Determination of Human Leukocyte Antigen (“HLA”) Binding

In this experiment, synthetic peptides corresponding to differentregions in either a wild-type or engineered ¹⁰Fn3 domain sequence wereevaluated in HLA binding assays. Similar HLA binding assays aredescribed in Reijonen H, Kwok W W, Use of HLA class II tetramers intracking antigen-specific T cells and mapping T-cell epitopes, Methods29(3):282-8 (2003). Each experimental peptide tested in the HLA bindingassay was either a wildtype ¹⁰Fn3 domain north pole loop (BC, DE or FGloop) peptide segment having additional amino acids flanking the N- andC-termini of each loop (SEQ ID NOs: 58, 51 and 52), a scaffold regionpeptide segment from a wildtype ¹⁰Fn3 domain that is positionedN-terminus to the BC loop (SEQ ID NO: 53) or a scaffold region peptidesegment from an engineered ¹⁰Fn3 domain that is positioned C-terminus tothe BC loop (SEQ ID NO: 54). The experimental peptides were solvated in100% DMSO at 50× the concentration desired in the assay. Each peptidewas then diluted into reaction buffer and titrated serially from 128 μMto 2 μM.

In the HLA binding assay, each of five different HLA allele molecules(either DRB*0101; DRB*0301; DRB*0401; DRB*0701 or DRB*1501 alleles) wereloaded separately into wells of a 96-well plate along with the unlabeledexperimental peptides and a europium-labeled control (competitor)peptide. The binding mixture was incubated in the wells for 24 hours soas to reach steady equilibrium. The HLA molecule complexes then werecaptured on an ELISA plate coated with anti-human HLA-DR antibody. BoundHLA-labeled control peptide was measured by time-resolved fluorescenceand assessed at 615 nm by a Wallac Victor3™ unit (Perkin-Elmer). Bindingof experimental peptides was expressed as the percent inhibition of thelabeled control peptide (experimental fluorescence/control fluorescencemultiplied by 100). From the percent inhibition of labeled controlpeptide at each concentration, IC₅₀ curves were derived for eachexperimental peptide against the five alleles tested. The results fromthese experiments are illustrated in FIG. 4.

As shown in FIG. 4, the BC loop peptide was observed to bind to four ofthe five alleles tested with high affinity suggesting it is animmunodominant sequence within the protein. By contrast, syntheticpeptides corresponding to the DE and FG loops bound fewer HLA allelesand with lower affinity, in general. The DE and FG loops are, therefore,not predicted to be immunodominant sequences within the wild-type ¹⁰Fn3protein.

As shown in FIG. 4, the scaffold region peptide of SEQ ID NO: 53 wasfound to bind to five of the HLA alleles with high affinity, while thescaffold region peptide of SEQ ID NO: 54 was found to bind to four ofthe HLA alleles with high affinity. These results suggest that thescaffold region flanking the BC loop of a ¹⁰Fn3 domain areimmunodominant regions. The underlined portions of the sequences of SEQID NOs: 53 and 54 as shown in FIG. 4 are predicted to be theimmunodominant portions of these sequences.

The BC loop, which was found to be an immunodominant loop, was furtherassessed using the same HLA binding assay. Specifically, three BC loopsequence variant peptides (SEQ ID NOs: 55-57) were examined using theassay and found to show almost identical patterns of strong binding tothe HLA alleles tested. The peptide sequence of SEQ ID NO: 55 is thehuman wildtype sequence and SEQ ID NOs: 56 and 57 are the BC loopregions from ¹⁰Fn3 domains that have been engineered to bind to twodifferent targets. Assuming that a similar binding pattern reflects ashared motif, the sequences were aligned to aid identification ofpotential anchor residues. Potential position 1 residues of thedifferent BC loop sequences tested are underlined:

(sequence position based on SEQ ID NO:1) 15       23      30 |        |       | PTSLLISW DAPAVTV RYYRITYG (SEQ ID NO: 55)PTSLLISW SARLKVA RYYRITYG (SEQ ID NO: 56)PTSLLISW RHPHFPT RYYRITYG (SEQ ID NO: 57)

The common sequence portions of these three peptides are the β-strand B,preceding the variable BC loop, and the β-strand C following the BCloop. Each of these portions has several hydrophobic residues (potentialposition 1 anchors), but those in 3-strand C do not have at least 8 moreresidues following, and therefore cannot be the anchor residues for MHCbinding. The hydrophobic residues in the variable BC loop are indifferent positions in the three peptides, making it unlikely that asingle 9-mer position with shared β-strand C residues can be anchored inthe BC loops. These results therefore suggest that β-strand B should beuseful for designing the peptide anchors.

The most likely positions for anchor residues appear to be “LLI”(positions 18-20 of SEQ ID NO: 1), as these include a stretch of fixedβ-strand B residues preceding the BC loop residues. If, for instance, a9-mer beginning at the first L is anchoring the peptide, the fourthposition is always an S, which is favorable for binding to many HLAalleles.

It should be noted that while many fully human sequences are displayedby MHC, the immune system recognizes them as “self” and does not mountan immune response. Further, in Cyno monkeys, which have the identicalsequence for ¹⁰Fn3, an immune response was generated upon administrationof various different ¹⁰Fn3 polypeptides, but no immune response wasgenerated upon the administration of wildtype ¹⁰Fn3. This indicates thatCyno monkeys recognize the human wildtype ¹⁰Fn3 sequence as being a“self” protein, to which an immune response need not be mounted.

B—in Silico Prediction of HLA Binding

HLA binding may be predicted in silico, for example, using EpiMatrix.EpiMatrix is a proprietary computer algorithm developed by EpiVax, whichis used to screen protein sequences for the presence of putative HLAbinding motifs. Input sequences are parsed into overlapping 9-mer frameswhere each frame overlaps the last by 8 amino acids. Each of theresulting frames is then scored for predicted binding affinity withrespect to a panel of eight common Class II HLA alleles (DRB1*0101,DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, andDRB1*1501). Raw scores are normalized against the scores of a largesample of randomly generated peptides. The resulting “Z” score isreported. Any 9-mer peptide with an EpiMatrix Z-score in excess of 1.64is considered a putative HLA binding motif.

Peptide epitopes from a ¹⁰Fn3 polypeptide having the amino acid sequenceset forth in SEQ ID NO: 61 were predicted using the EpiMatrix algorithm.Results showed the BC, DE and FG loops of the ¹⁰Fn3 polypeptide haveZ-scores of 21.3, 0.9 and 1.1, respectively.

The above in vitro and in silico HLA binding results suggest that theβ-strand B/BC loop segment may be a ‘hot spot’ for the purposes of HLAbinding. The β-strand B/BC loop segment may be anchored strongly to MHCmolecules by the amino acids in β-strand B, such that at least somevariants in BC loop sequences make little difference in the binding toHLA. The strong anchor may make it difficult to deimmunize this segment.However, if the entire stretch is a wildtype sequence, it should berecognized as self by the immune system, and initiate no immuneresponse. Therefore, ¹⁰Fn3 domain polypeptide libraries may be designedto have lower immunogenicity by leaving the BC loop as wildtype, or atthe very least, by leaving the residues from D23 to T28 of SEQ ID NO: 1or 6 as wildtype in order to retain as wildtype any 9-mer peptideanchored at L18, L19, or 120. Examples of libraries in which all or asubstantial portion of the BC loop was left wild-type are shown in FIG.9A. Examples of libraries in which varying portions of the N-terminalregions of the BC loop are left as wild-type are shown in FIG. 9B.Specific examples of ¹⁰Fn3 binders in which the residues correspondingto D23 to T28 of SEQ ID NO: 1 were left wildtype are provided in Example3. Alternatively, if the BC loop is modified, reduced immunogenicity maybe achieved by destroying the strong anchor in this region, i.e., bymaking modifications in β-strand B, in addition to the modifications inthe BC loop. Examples of libraries in which the anchor is removedinclude libraries in which positions L19 and/or S21 have beendiversified thereby increasing the likelihood that members of thelibrary will be missing the anchor residues. Examples of libraries inwhich the anchor residues are removed are shown in FIG. 9C.

Example 3. Generation of West Side Binders

Libraries of West Side (“WS”) binder polypeptides comprising a modified¹⁰Fn3 domain were screened using mRNA display (Xu et al Chem Biol. 2002August; 9(8):933-42) for binding to murine IL-17, murine DLL4 or humanPXR as targets. The WS binders were designed such that the BC loopsequence was left as wildtype. The WS1 library design (see FIG. 9A) wasused to identify binders to the target human PXR and the WS-LI1 librarydesign (see FIG. 9A) was used to identify binders to the targets murineIL-17 or murine DLL4. Target binding was monitored by qPCR andpopulations were cloned and expressed in E. coli when a specific bindingsignal was observed.

Example 4. Disruption of DLL4 and Notch 1 Interaction by WS-LI1 BindersCapable of Binding Murine DLL4

DLL4 is a ligand for the Notch 1 protein. The ability of ¹⁰Fn3polypeptides having the WS-LI1 design to disrupt the interaction betweenNotch 1 and murine DLL4 was assessed by employing a competitive Biacoreexperiment. Approximately 4500 RU of Notch1-Fc was immobilized on a CM5Biacore chip. 2 μM of WS-LI1 binders were equilibrated with 20 nM murineDLL4 in HBSP buffer and 5 mM CaCl₂ along with a control in which nopolypeptide was added. Each sample was flowed over the chip and bindingof murine DLL4 was compared to the control in which no WS-LI1polypeptide was added, so that a reduction in signal corresponded toinhibition of the Notch 1:murine DLL4 interaction. Between each samplerun, the chip was regenerated with two 30-second washes in HBSP pH 7.4and 50 mM EDTA. The results of this experiment are shown in FIG. 5. Thepolypeptides having the sequence of SEQ ID NO: 3 and 4 were each foundto result in 100% competition of the interaction between Notch1 andmurine DLL4. The polypeptides having the sequence of SEQ ID NO: 5 wereable to induce 75% competition of the interaction between Notch 1 andmurine DLL4.

Example 5. Size Exclusion Chromatography Analysis of WS-LI1 BindersCapable of Binding Murine DLL4

Size exclusion chromatography was utilized to demonstrate that thecompetition results observed in Example 4 were due to monomeric forms ofthe WS-LI1 binders tested. The WS-LI1 binders having amino acidsequences of either SEQ ID NO: 3 or 4 were predominantly monomeric,whereas the WS-LI1 binder having the amino acid sequence of SEQ ID NO: 5contained a mixture of monomeric and aggregated proteins. The resultsfrom this experiment are shown in FIG. 6 and illustrate that the WS-LI1binders tested in Example 4 were acting as a monomeric species,suggesting that the binders exist as stable, well-folded polypeptides.

Example 6. Stability of WS-LI1 Binders Capable of Binding Murine DLL4

Stability of the polypeptides described in Example 4 was assessed by athermal shift fluorescence-based assay (TSF). The polypeptide having theamino acid sequence of SEQ ID NO: 3 had a transition at 59° C. Thepolypeptide having the amino acid sequence of SEQ ID NO: 4 had atransition at 49° C. No transition was observed for the polypeptidehaving the amino acid sequence of SEQ ID NO: 5.

Example 7. Disruption of the Interaction Between Murine IL-17 and MurineIL-17RA by WS-LI1 Binders Capable of Binding Murine IL-17

IL-17 is a ligand for the IL-17 receptor A protein, IL-17RA. The abilityof ¹⁰Fn3 domains having the WS-LI1 design to disrupt the interactionbetween murine IL-17 and murine IL-17RA was assessed by employing acompetitive Alphascreen experiment. Streptavidin donor beads, anti-humanIgG acceptor beads, 1.5 nM murine IL-17RA-Fc and 2.5 nM biotinylatedmurine IL-17 were combined according to the manufacturer's instructionsto give a robust Alphascreen signal. Polypeptides having the sequencesof either SEQ ID NO: 45, 46 or 47 were assessed for their ability toinhibit this signal when added to the mixture at 1 μM concentrations.The polypeptide having the sequence of SEQ ID NO: 45 caused an 83%inhibition, the polypeptide having the sequence of SEQ ID NO: 46 causeda 94% inhibition and the polypeptide having the sequence of SEQ ID NO:47 caused an 81% inhibition of the Alphascreen signal. These resultsdemonstrate that the WS-LI1 library design produced ¹⁰Fn3 domainscapable of binding IL-17 and effectively inhibiting the interactionbetween murine IL-17 and its receptor.

Example 8. Size Exclusion Chromatography Analysis of WS-LI1 BindersCapable of Binding Murine IL-17

Size exclusion chromatography was utilized to demonstrate that thecompetition results observed in Example 7 were due to monomeric forms ofthe WS-LI1 binders tested. The WS-LI1 binders having amino acidsequences of either SEQ ID NO: 45, 46 or 47 were predominantlymonomeric. The results from this experiment are shown in FIG. 7 andillustrate that the WS-LI1 binders tested in Example 7 were acting as amonomeric species, suggesting that the binders exist as stable,well-folded polypeptides.

Example 9. Stability of WS-LI1 Binders Capable of Binding Murine IL-17

Stability of the polypeptides described in Example 7 was assessed byTSF. The polypeptide having the amino acid sequence of SEQ ID NO: 45 hada transition at 51° C. The polypeptide having the amino acid sequence ofSEQ ID NO: 46 had a transition at 60° C. No transition was observed forthe polypeptide having the amino acid sequence of SEQ ID NO: 47.

Example 10. Characterization of Binding Properties of WS1 BindersCapable of Binding Human PXR

WS1 binders that were capable of binding to PXR were characterized usinga Biacore binding assay and GST-tagged PXR. 14000 RU of anti-GSTantibody was immobilized on a Biacore chip and GST-PXR was captured byflowing a 50 nM solution over the chip for 3 min at a rate of 5 μL/min.WS1 binders were flowed over the chip at 0.5-2 μM concentration toobserve binding relative to a control that lacked PXR. The chip wasstripped between each run using two 30 second washes with 10 mM glycine,pH 2.0 and fresh GST-PXR was captured. Under these conditions, 146 RU ofWS1 binders having the amino acid sequence of SEQ ID NO: 48 bound theGST-PXR, and 81 RU of WS1 binders having the amino acid sequence of SEQID NO: 49 bound the GST-PXR. These results demonstrate that the WS1binders having the amino acid sequence of either SEQ ID NO: 48 or 49 arecapable of binding GST-PXR.

Example 11. Size Exclusion Chromatography Analysis of WS1 BindersCapable of Binding Human PXR

Size exclusion chromatography was utilized to demonstrate that thebinding results observed in Example 10 were due to monomeric forms ofthe WS1 binders tested. The WS1 binders having amino acid sequences ofeither SEQ ID NO: 48 or 49 were predominantly monomeric. The resultsfrom this experiment are shown in FIG. 8 and illustrate that the WS1binders tested in Example 10 were acting as a monomeric species,suggesting that the binders exist as stable, well-folded polypeptides.

Example 12. Sequences and Binding Characteristics of 10Fn3 PolypeptidesBinding to Human PXR

This Example describes 6 additional ¹⁰Fn3 polypeptides that bind tohuman PXR ligand binding domain (LBD). It also provides bindingcharacteristics of these 6 polypeptides as well as the two that aredescribed in Example 10 (and having SEQ ID NOs: 48 and 49).

¹⁰Fn3 polypeptides having the amino acid sequences set forth in FIG. 10were identified by screening various libraries. For example, Adnectin-1was isolated from an NP1 library (see FIG. 9C). The ¹⁰Fn3 polypeptideswere synthesized as follows. Nucleic acids encoding the ¹⁰Fn3polypeptides were cloned in pET9D vector and then expressed inEscherichia coli at 20° C. The lysates were purified in a single stepusing Ni-agarose affinity chromatography.

The K_(D) values for the ¹⁰Fn3 polypeptides were determined by surfacePlasmon resonance (SPR) on a Biacore T100 instrument (GE Healthcare), byinjecting a concentration series of the ¹⁰Fn3 polypeptides over humanPXR-GST (Invitrogen) that had been captured on chip immobilized with ananti-GST antibody (GE Healthcare). Regeneration of the chip surfacebetween kinetic cycles was performed using 10 mM Glycine, pH 2. Kineticparameters for both were calculated using Biacore T100 software. Theresults are set forth in Table 1.

TABLE 1 Binding characteristics of Adnectins-1 to -8 to human PXRAdnectin ka (1/Ms) kd (1/s) KD (nM) Adnectin-1 1.04E+05 ± 1.20E+041.19E−03 ± 3.46E−05 11.4 ± 1 Adnectin-2 1.62E+05 ± 5.37E+03 4.58E−03 ±3.53E−04 97.8 ± 3.8 Adnectin-3 1.62E+04 ± 6.75E+04 3.78E−04 ± 2.23E−05 2.5 ± 0.9 Adnectin-4 2.15E+04 ± 5.30E+02 1.68E−04  7.8 Adnectin-55.09E+05 ± 1.07E+05 2.70E−03 ± 2.36E−04  5.4 ± 0.7 Adnectin-6 6.80E+05 ±1.69E+05 1.26E−03 ± 1.72E−04  1.9 ± 0.2 Adnectin-7 2.88E+05 ± 2.38E+048.82E−05 ± 1.15E−06 0.31 ± 0.03 Adnectin-8 4.17E+04 ± 8.22E+03 1.82E−04± 2.78E−05  4.4 ± 0.2Adnectin-1 was effectively used as a co-crystallization chaperone ofhuman PXR with a small molecule, and shown to bind to the ligand bindingdomain of PXR. The X-ray data provided information on the interactionbetween the small molecule and the PXR ligand binding domain.

Example 13. Lack of RGD in FG Loop Prevents Binding of ¹⁰Fn3Polypeptides to Fibronectin and Vitronectin

Recombinant human integrin αV33 (R&D Systems, Minneapolis Minn.) wasdiluted to 40 ug/mL in Acetate buffer pH5.0 (GE Healthcare, PiscatawayN.J.), and. then immobilized on a CM7 chip (GE Healthcare) usingstandard amine coupling techniques. 500 nM fibronectin (RocheDiagnostics, Indianapolis, Ind.) and vitronectin (R&D Systems) and 5 μMof either non-binding control ¹⁰Fn3 molecule (consisting of SEQ ID NO: 6with an additional MG at the N-terminus and with a single amino acidsubstitution that changes RGD to RGE) or targeted ¹⁰Fn3 molecules(having a mutated FG loop that does not contain an RGD motif) wereflowed over the top of the immobilized integrin. Binding RU wascollected at the end of the sample injection. The results indicate thatthe lack of RGD in the FG loop results in abolishing binding of ¹⁰Fn3molecules to fibronectin and vitronectin.

Example 14. Characteristics of Molecules Obtained from Various Libraries

This Example shows various characteristics of molecules obtained from 11different libraries. The following libraries were made:

-   -   LI-1 library, which is a mixture of libraries LI-1 (a), LI-1(b)        and LI-1(c), comprising ¹⁰Fn3 molecules having the amino acid        sequences that are provided in FIG. 9B. The amino acid residues        that are underlined are those that were varied to any amino acid        by substitution. Residues that are underlined may be the        wild-type residues;    -   LI-3 library comprising ¹⁰Fn3 molecules having the amino acid        sequence that is provided in FIG. 9B (“LI-3(b)), wherein the        amino acid residues that are boxed were varied by substitution        to any amino acid or deletion or addition. Residues that are        boxed may be the wild-type residues;    -   WS-LI1 library, comprising ¹⁰Fn3 molecules having the amino acid        sequence set forth in FIG. 9A, wherein the amino acid residues        that are boxed were varied by substitution to any amino acid or        deletion or addition. Residues that are boxed may be the        wild-type residues;    -   LI-S9 library, comprising ¹⁰Fn3 molecules having the amino acid        sequence set forth in FIG. 9A, wherein the boxed residues were        varied by amino acid substitution, addition or deletion, the        underlined residues were varied by substitution, and the        highlighted residue is changed to an E. Residues that are boxed        or underlined may be the wild-type residues;    -   LI-S8 library, comprising ¹⁰Fn3n3 molecules having the amino        acid sequence set forth in FIG. 9A, wherein the boxed residues        were varied by substitution, addition or deletion, and the        highlighted residue is changed to an E. Residues that are boxed        may be the wild-type residues;    -   NP4 FG library, comprising ¹⁰Fn3 molecules having the amino acid        sequence set forth in FIG. 9C, wherein the boxed residues were        varied by amino acid substitution, addition or deletion, and the        underlined residues were varied by substitution. Residues that        are boxed or underlined may be the wild-type residues. This        library corresponds to the NP4 library, wherein only residues in        FG, F and G were varied.    -   WS2′ CD library, comprising ¹⁰Fn3 molecules having the amino        acid sequence set forth in FIG. 9A, wherein the boxed residues        were varied by amino acid substitution, addition or deletion,        and the underlined residues were varied by substitution.        Residues that are boxed or underlined may be the wild-type        residues. This library corresponds to the WS2′ library, wherein        only residues in CD, C and D were varied;    -   NP1′ library, comprising ¹⁰Fn3 molecules having the amino acid        sequence set forth in FIG. 9C, wherein the boxed residues were        varied by amino acid substitution, addition or deletion, and the        underlined residues were varied by substitution. Residues that        are boxed or underlined may be the wild-type residues;    -   NP4 library, comprising ¹Fn3 molecules having the amino acid        sequence set forth in FIG. 9C, wherein the boxed residues were        varied by amino acid substitution, addition or deletion, and the        underlined residues were varied by substitution. Residues that        are boxed or underlined may be the wild-type residues; and    -   WS2′ library, comprising ¹Fn3 molecules having the amino acid        sequence set forth in FIG. 9A, wherein the boxed residues were        varied by amino acid substitution, addition or deletion, and the        underlined residues were varied by substitution. Residues that        are boxed or underlined may be the wild-type residues.

Clones from each of the libraries were expressed and purified. Theresulting ¹⁰Fn3 molecules were run on the Perkin Elmer LabChip GX withthe low molecular weight protein ladder per the manufacturers'recommendations. The concentrations of the ¹Fn3 molecules wereextrapolated from a standard curve of SGE also run on the LabChip GX.Samples with concentrations higher than that of the standard curve wereclassified as off-curve-high (OCH). The percent of OCH wells and theaverage concentration for each library was determined. The resultsindicate that the fraction of OCH wells and average concentration ofmeasurable samples follow similar patterns.

The resistance to aggregation of ¹Fn3 molecules from each of thelibraries was also tested. Between 19-200 clones from each library wereassayed for aggregation and retention time by size exclusionchromatography (SEC) according to manufacturers' recommendations. TheSEC chromatogram was integrated for each clone and evaluated for peaksthat eluted between the times of a 17 kDa and 1.3 kDa standard. Cloneshaving greater than 90% area in this retention time window received anSEC score of 1, 50%-90% a score of 2, 5%-50% a score of 3, and less than5% a score of 4. Fraction of SEC score 1 or 2 is measure of resistanceto aggregation and fraction SEC 4 is a measure of load on SEC screening.The results indicate that fraction of SEC 1 or 2 and fraction of SEC 4follow approximately inverse patterns.

Target binding of clones from each library was determined by ELISA. Theproteins that the clones were directed against were coated at 2.5 μg/mLon a Nunc Maxisorp Plate. Plates were blocked with Casein(ThermoScientific #37532) and then incubated with the ¹⁰Fn3 molecules.Binding clones were detected through a His-tag using an HRP labeledanti-His antibody (R&D Systems # mAb050H). Hits were determined asanything which gave a signal greater than 2× that of blank wells. Thefraction of hits is a measure of load on screening. All libraries had atleast an expected number of hits.

The existence of multiple epitopes to which members of each librarybound was also determined. For three targets this involved competitiveAlphascreen assays as referenced in Example 7. For these targets,multiple epitope coverage was proven by the existence of competitors andnon-competitors from proven target binders. For one multi-domain target,multiple epitope binding was demonstrated using an ELISA withrecombinant versions of the individual subdomains. The fraction oftargets for which multiple epitopes are found is a measure ofprobability of finding diverse biological activity in a library. Theresults of multiple epitope coverage for multiple targets with thedifferent libraries indicate diversity of blocking.

INCORPORATION BY REFERENCE

All documents and references, including patent documents and websites,described herein are individually incorporated by reference to into thisdocument to the same extent as if there were written in this document infull or in part.

1. A polypeptide comprising a tenth fibronectin type 3 (¹⁰Fn3) domain,wherein the ¹⁰Fn3 domain comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 76-87, 118-121 and 128-131, andwherein at least 50% of amino acids x are not the amino acid at thecorresponding position in SEQ ID NO: 1 or
 6. 2. The polypeptide of claim1, wherein the ¹⁰Fn3 domain binds specifically to a target with a Kd ofless than 500 nM that is not bound by a ¹⁰Fn3 domain having an aminoacid sequence consisting of SEQ ID NO: 1 or
 6. 3. The polypeptide ofclaim 1, wherein the ¹⁰Fn3 domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 76-87, 118-121 and128-131, wherein each x amino acid residue is any amino acid residueexcept the amino acid at the corresponding position in SEQ ID NO: 1 or6.
 4. The polypeptide of claim 1, wherein the amino acid sequence of the¹⁰Fn3 domain that is located outside of the variable regions is at least90% identical to the corresponding regions in SEQ ID NO: 1 or
 6. 5. Thepolypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises an amino acidsequence that differs from SEQ ID NO: 1 or 6 in at most 20, 15 or 10amino acids.
 6. The polypeptide of claim 1, wherein the ¹⁰Fn3 domaindoes not comprise any other amino acid variation relative to SEQ ID NO:1 or
 6. 7. The polypeptide of claim 1, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence that is at least 90% identical to SEQID NO: 1 or
 6. 8. A library comprising a plurality of polypeptidescomprising a ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 76-87,118-121 and 128-131, and wherein at least 50% of amino acids x are notthe amino acid at the corresponding position in SEQ ID NO: 1 or
 6. 9.The library of claim 8, wherein each x amino acid residue is any aminoacid residue except the amino acid at the corresponding position in SEQID NO: 1 or
 6. 10. The library of claim 8, wherein the library comprisesat least 10⁵ polypeptides each comprising a different ¹⁰Fn3 amino acidsequence.
 11. A method for identifying a polypeptide that binds to atarget comprising screening the library of claim 8 to identify apolypeptide that binds to the target.
 12. An isolated polypeptideidentified by the method of claim
 11. 13. A nucleic acid encoding thepolypeptide of claim
 1. 14. A vector comprising the nucleic acid ofclaim
 13. 15. A host cell expressing the nucleic acid of claim 13.